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Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Many strands of research by different groups, starting from teratocarcinomas in the laboratory mouse, later moving the corresponding human tumors, contributed to the isolation and description of human pluripotent stem cells (PSCs). In this review, I highlight the contributions from my own research, particularly at the Wistar Institute during the 1980s, when with my colleagues we characterized one of the first clonal lines of pluripotent human embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, and identified key features including cell surface antigen markers that have since found a place in the study and exploitation of human PSC. Much of this research depended upon close teamwork with colleagues, many in other laboratories, who contributed different expertise and experience. It was also often driven by circumstance and chance rather than pursuit of a grand design. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 immunemediated neurological syndromes, and negative testing in established routine laboratories, opening new perspectives in establishing a diagnosis of such complex diseases. [ABSTRACT FROM AUTHOR]

Introduction: Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes. Results and discussion: Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes. [ABSTRACT FROM AUTHOR]

Organoids are quickly becoming an accepted model for understanding human biology and disease. Pluripotent stem cells (PSC) provide a starting point for many organs and enable modeling of the embryonic development and maturation of such organs. The foundation of PSC‐derived organoids can be found in elegant developmental studies demonstrating the remarkable ability of immature cells to undergo histogenesis even when taken out of the embryo context. PSC‐organoids are an evolution of earlier methods such as embryoid bodies, taken to a new level with finer control and in some cases going beyond tissue histogenesis to organ‐like morphogenesis. But many of the discoveries that led to organoids were not necessarily planned, but rather the result of inquisitive minds with freedom to explore. Protecting such curiosity‐led research through flexible funding will be important going forward if we are to see further ground‐breaking discoveries. [ABSTRACT FROM AUTHOR]

Despite significant advances in the prevention, diagnosis, and treatment of cardiac diseases, this condition is still considered one of the major challenges facing the global healthcare system. According to the World Health Organization (WHO) statistics, cardiovascular diseases (CVD) are among the leading causes of mortality worldwide. It is predicted that in the future, due to population aging, urbanization, and lifestyle changes, the mortality rate from cardiac diseases will increase. Current pharmacological approaches only extend the lifespan of patients without providing cardiac tissue repair. In the advanced stages of these diseases, apart from heart transplantation, there is no curative treatment available. However, due to limited suitable donors and post-operative risks and complications such as graft rejection, primary graft failure, and common infections after heart transplant, transplantation is not always feasible. Stem cells have created a wide range of hope in the world since their discovery. With increased research on stem cells and cell therapy in recent decades, researchers have found that utilizing this innovative therapeutic approach can increase lifespan and improve patient outcomes. Various countries have invested millions of dollars in the application of stem cells for treating various diseases in recent years. Embryonic stem cells (ESCs) have great differentiation potential, but their medical application faces severe ethical and religious constraints. The identification of potent induced pluripotent stem cells has revolutionized regenerative medicine. These cells possess all the capabilities of embryonic stem cells and can easily differentiate into cardiac myocytes. Furthermore, the paracrine effects of induced pluripotent stem cells (iPSCs) and extracellular vesicles (EVs) secreted from these cells in regenerative medicine have garnered significant attention. Among extracellular secretory vesicles, researchers have focused more on exosomes. Exosomes are vesicles ranging from 30 to 100 nanometers that have a plasma membrane-like topology and can enter target cells and deliver their cargo. Cardiac patches are a novel achievement resulting from the integration of tissue engineering and cell sciences and are used to improve cardiac injuries. Cardiac patches consist of a natural or synthetic scaffold designed to support and restore myocardial tissue following injury, and they are implanted into the heart tissue. The primary approach to using cardiac patches is to provide physical support to damaged heart tissue. Nowadays, the combination of cardiomyocytes derived from potent induced pluripotent stem cells along with biocompatible materials and growth factors has created specialized cardiac patches capable of precise delivery of many cells and minimizing cellular damages in vivo conditions. This review article aims to explore the latest advances in cardiac regenerative medicine through the use of cardiac patches, induced pluripotent stem cells, and exosomes derived from them; as well as to assess the challenges ahead in cardiac regenerative medicine using the mentioned items to alleviate and improve tissue damage to the heart, and ultimately provide an overview of new perspectives for future research in cardiac regenerative medicine. [ABSTRACT FROM AUTHOR]

Background Fabry disease (FD) is a rare X-linked inherited disease caused by mutations in the galactosidase alpha (GLA) gene. We established a cohort of FD patients and performed whole-exome sequencing to identify some novel mutations. Aim The aim of this study is to investigate the etiology of the novel mutation (c.72G > A, p. Trp24*)in the GLA gene in affected patients by using induced pluripotent stem cells (iPSCs) as a valuable tool. Methods We explored the clinical implications of this proband and examined the deleteriousness and conservation of the mutation site through bioinformatics analysis. Simultaneously, we collected the peripheral blood mononuclear cells of the affected patient, then reprogrammed them into iPSCs and assessed their enzymatic activity to confirm the function of lysosomal enzyme α-galactosidase A (α-Gal A). Results Clinical examination of the patient demonstrated a classical FD, such as neuropathic pain, gastrointestinal disorders, deficiency of α-Gal A activity and accumulation of Lyso-Gb-3. The novel mutation located on the N-terminal region, leading to a truncation of the protein and remaining only 24 amino acids. The α-Gal A activity of the patient-specific iPSC (iPS-FD) was significantly lower (60%) than that of normal iPSCs derived from healthy donors (iPS-B1). Conclusion This work not only elucidated the etiology of novel mutations in affected patients but also highlighted the utility of iPSCs as a valuable tool for clarifying the molecular mechanisms and providing new insights into the therapy of FD. [ABSTRACT FROM AUTHOR]

Artificial oocytes were used of different mammalian species for repairing reproductive dysfunctions. Artificial oocytes are those created in vitro using enucleated germinal vesicle, metaphase I and metaphase II oocytes followed by embryonic cells, somatic cells, pluripotent stem cells and germ cells nuclear transfer. Artificial oocytes are under development, they have the potential to revolutionize reproduction in mammalian species. The potential applications of artificial oocytes include 1) treatment of infertility as premature ovarian failure, ovarian cancer, or other conditions that damage the ovaries, 2) preservation of fertility, 3) creating cloned and transgenic animals. W hile artificial oocytes have the potential to revolutionize reproductive medicine, there are still a number of challenges that need to be addressed before they can be effectively and safely used in humans including the adapting efficient and safe enucleation techniques. Furthermore, cellular reprogramming is the biggest obstacle for creating artificial oocytes in addition to activation procedures to ensure that artificial oocytes are genetically and epigenetically normal and producing healthy offspring. Several studies are designed to overcome these challenges and there is significant progresses being made. Offspring are obtained in several animals species whereas developed blastocysts were obtained in humans. This review is developed and implemented to discuss the progress in creation of "artificial oocytes" in addition to the factors affecting the developmental competence of reconstructed oocytes. [ABSTRACT FROM AUTHOR]

Kidney organoids -- 3D representations of kidneys made either from pluripotent or tissue stem cells -- have been available for well over a decade. Their application could confer notable benefits over longstanding in vivo approaches with the potential for clinically aligned human cells and reduced ethical burdens. They been used, at a proof-of-concept level, in development in disease modeling (including with patient-derived stem cells), and in screening drugs for efficacy/toxicity. They differ from real kidneys: they represent only foetal-stage tissue, in their simplest forms they lack organ-scale anatomical organization, they lack a properly arranged vascular system, and include non-renal cells. Cell specificity may be improved by better techniques for differentiation and/or sorting. Sequential assembly techniques that mimic the sequence of natural development, and localized sources of differentiation-inducing signals, improve organ-scale anatomy. Organotypic vascularization remains a challenge: capillaries are easy, but the large vessels that should serve them are absent from organoids and, even in cultured real kidneys, these large vessels do not survive without blood flow. Transplantation of organoids into hosts results in their being vascularized (though probably not organotypically) and in some renal function. It will be important to transplant more advanced organoids, with a urine exit, in the near future to assess function more stringently. Transplantation of human foetal kidneys, followed by nephrectomy of host kidneys, keeps rats alive for many weeks, raising hope that, if organoids can be produced even to the limited size and complexity of foetal kidneys, they may one day be useful in renal replacement. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channel 1 (ASIC1) is critical in acidotoxicity and significantly contributes to neuronal death in cerebral stroke. Pharmacological inhibition of ASIC1 has been shown to reduce neuronal death. However, the potential of utilizing exosomes derived from pluripotent stem cells to achieve inhibition of Asic1 remains to be explored. Developing qualified exosome products with precise and potent active ingredients suitable for clinical application is also ongoing. Here, we adopt small RNA-seq to interrogate the miRNA contents in exosomes of pluripotent stem cell induced mesenchymal stem cell (iMSC). RNA-seq was used to compare the oxygen-glucose deprivation–damaged neurons before and after the delivery of exosomes. We used Western blot to quantify the Asic1 protein abundance in neurons before and after exosome treatment. An in vivo test on rats validated the neuroprotective effect of iMSC-derived exosome and its active potent miRNA hsa-mir125b-5p. We demonstrate that pluripotent stem cell–derived iMSCs produce exosomes with consistent miRNA contents and sustained expression. These exosomes efficiently rescue injured neurons, alleviate the pathological burden, and restore neuron function in rats under oxygen-glucose deprivation stress. Furthermore, we identify hsa-mir-125b-5p as the active component responsible for inhibiting the Asic1a protein and protecting neurons. We validated a novel therapeutic strategy to enhance acidosis resilience in cerebral stroke by utilizing exosomes derived from pluripotent stem cells with specific miRNA content. This holds promise for cerebral stroke treatment with the potential to reduce neuronal damage and improve clinical patient outcomes. [ABSTRACT FROM AUTHOR]

The epididymis, a key reproductive organ, is crucial for sperm concentration, maturation, and storage. Despite a comprehensive understanding of many of its functions, several aspects of the complex processes within the epididymis remain obscure. Dysfunction in this organ is intricately connected to the formation of the microenvironment, disruptions in sperm maturation, and the progression of male infertility. Thus, elucidating the functional mechanisms of the epididymal epithelium is imperative. Given the variety of cell types present within the epididymal epithelium, utilizing a three-dimensional (3D) in vitro model provides a holistic and practical framework for exploring the multifaceted roles of the epididymis. Organoid cell culture, involving the co-cultivation of pluripotent or adult stem cells with growth factors on artificial matrix scaffolds, effectively recreates the in vivo cell growth microenvironment, thereby offering a promising avenue for studying the epididymis. The field of epididymal organoids is relatively new, with few studies focusing on their formation and even fewer detailing the generation of organoids that exhibit epididymis-specific structures and functions. Ongoing challenges in both clinical applications and mechanistic studies underscore the importance of this research. This review summarizes the established methodologies for inducing the in vitro cultivation of epididymal cells, outlines the various approaches for the development of epididymal organoids, and explores their potential applications in the field of male reproductive biology. [ABSTRACT FROM AUTHOR]

Urine-derived stem cells (USCs) are a promising source of adult stem cells for cell therapy, drug testing for renal toxicity, and biomarker discovery in kidney disease. USCs can be non-invasively isolated from the donor's own urine, making them a good candidate for precision medicine. Preclinical studies have shown that USCs and exosomes can be used to repair damaged tissues (particularly urinary tract tissues and organs, including the kidneys), to test the toxicity of new drugs, and to identify biomarkers for kidney disease. Further research is needed to fully understand the potential of USCs and to develop safe and effective ways to explore their full potential in precision medicine. Urine-derived stem cells (USCs) are a promising source of stem cells for cell therapy, renal toxicity drug testing, and renal disease biomarker discovery. Patients' own USCs can be used for precision medicine. In this review we first describe the isolation and characterization of USCs. We then discuss preclinical studies investigating the use of USCs in cell therapy, exploring the utility of USCs and USC-derived induced pluripotent stem cells (u-iPSCs) in drug toxicity testing, and investigating the use of USCs as biomarkers for renal disease diagnosis. Finally, we discuss the challenges of using USCs in these applications and provide insights into future research directions. USCs are a promising tool for advancing renal therapy, drug testing, and biomarker discovery. Further research is needed to explore their potential. [ABSTRACT FROM AUTHOR]

Derivation of hypoimmunogenic human cells from genetically manipulated pluripotent stem cells holds great promise for future transplantation medicine and adoptive immunotherapy. Disruption of beta-2-microglobulin (B2M) in pluripotent stem cells followed by differentiation into specialized cell types is a promising approach to derive hypoimmunogenic cells. Given the attractive features of CRISPR/Cas9-based gene editing tool and baculoviral delivery system, baculovirus can deliver CRISPR/Cas9 components for site-specific gene editing of B2M. Herein, we report the development of a baculoviral CRISPR/Cas9 vector system for the B2M locus disruption in human cells. When tested in human embryonic stem cells (hESCs), the B2M gene knockdown/out was successfully achieved, leading to the stable down-regulation of human leukocyte antigen class I expression on the cell surface. Fibroblasts derived from the B2M gene-disrupted hESCs were then used as stimulator cells in the co-cultures with human peripheral blood mononuclear cells. These fibroblasts triggered significantly reduced alloimmune responses as assessed by sensitive Elispot assays. The B2M-negative hESCs maintained the pluripotency and the ability to differentiate into three germ lineages in vitro and in vivo. These findings demonstrated the feasibility of using the baculoviral-CRISPR/Cas9 system to establish B2M-disrupted pluripotent stem cells. B2M knockdown/out sufficiently leads to hypoimmunogenic conditions, thereby supporting the potential use of B2M-negative cells as universal donor cells for allogeneic cell therapy. [ABSTRACT FROM AUTHOR]

Primary ovarian insufficiency (POI) in younger women (under 40) manifests as irregular periods, high follicle-stimulating hormone (FSH), and low estradiol (E2), often triggered by chemotherapy. Though mesenchymal stem cell (MSC) therapy shows promise in treating POI, its exact mechanism remains unclear. This study reveals that human umbilical cord-derived MSCs (hUC-MSCs) can protect ovarian granulosa cells (GCs) from cyclophosphamide (CTX)-induced ferroptosis, a form of cell death driven by iron accumulation. CTX, commonly used to induce POI animal model, triggered ferroptosis in GCs, while hUC-MSCs treatment mitigated this effect, both in vivo and in vitro. Further investigations using ferroptosis and autophagy inhibitors suggest that hUC-MSCs act by suppressing ferroptosis in GCs. Interestingly, hUC-MSCs activate a protective antioxidant pathway in GCs via NRF2, a stress-response regulator. Overall, our findings suggest that hUC-MSCs improve ovarian function in CTX-induced POI by reducing ferroptosis in GCs. This study not only clarifies the mechanism behind the benefits of hUC-MSCs but also strengthens the case for their clinical use in treating POI. Additionally, it opens up a new avenue for protecting ovaries from chemotherapy-induced damage by regulating ferroptosis. [Display omitted] • hUC-MSCs effectively ameliorate ovarian dysfunction in CTX-induced POI model. • hUC-MSCs can reverse the iron overload and ferritinophagy in POI model and KGN cells. • hUC-MSCs can inhibit ferritinophagy-mediated ferroptosis in primary GCs of POI model and CTX-induced KGN cells. • NRF2 plays a key role in hUC-MSCs ameliorating ferritinophagy-mediated ferroptosis caused in POI model. [ABSTRACT FROM AUTHOR]

Retinal degeneration (RD) is a leading cause of blindness worldwide and includes conditions such as retinitis pigmentosa (RP), age-related macular degeneration (AMD), and Stargardt's disease (STGD). These diseases result in the permanent loss of vision due to the progressive and irreversible degeneration of retinal cells, including photoreceptors (PR) and the retinal pigment epithelium (RPE). The adult human retina has limited abilities to regenerate and repair itself, making it challenging to achieve complete self-replenishment and functional repair of retinal cells. Currently, there is no effective clinical treatment for RD. Stem cell therapy, which involves transplanting exogenous stem cells such as retinal progenitor cells (RPCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs), or activating endogenous stem cells like Müller Glia (MG) cells, holds great promise for regenerating and repairing retinal cells in the treatment of RD. Several preclinical and clinical studies have shown the potential of stem cell-based therapies for RD. However, the clinical translation of these therapies for the reconstruction of substantial vision still faces significant challenges. This review provides a comprehensive overview of stem/progenitor cell-based therapy strategies for RD, summarizes recent advances in preclinical studies and clinical trials, and highlights the major challenges in using stem/progenitor cell-based therapies for RD. [ABSTRACT FROM AUTHOR]

Non-coding RNA has many types which has rich functions and plays an important role in the study of basic molecular mechanisms. Many non-coding RNA have important implications for pluripotent stem cells and embryonic stem cells. It has been found to affect the self-renewal and osteogenesis of many types of stem cells. They have also been found to regulate stem cell proliferation and induct bone differentiation. Periodontal ligament stem cells are essential for the regeneration of periodontal tissue. In recent years, in the field of stomatology, studies have found that many non-coding RNA also have significant regulatory effects on the proliferation and differentiation of periodontal stem cells and may become potential therapeutic targets for many common periodontal diseases such as periodontitis, bone/tooth/soft tissue loss and orthodontic treatment. Therefore, we summarized the current research status of non-coding RNA in the field of molecular mechanism of periodontal ligament stem cells and prospected its future progress. [ABSTRACT FROM AUTHOR]

Malfunction in spermatogenesis due to genetic diseases, trauma, congenital disorders or gonadotoxic treatments results in infertility in approximately 7% of males. The behavior of spermatogonial stem cells (SSCs) within three-dimensional, multifactorial, and dynamic microenvironment implicates a niche that serves as a repository for fertility, since can serve as a source of mature and functional male germ cells. Current protocols enable reprogramming of mature somatic cells into induced pluripotent stem cells (iPSCs) and their limited differentiation to SSCs within the range of 0–5%. However, the resulting human iPSC-derived haploid spermatogenic germ cell yield in terms of number and functionality is currently insufficient for transfer to infertility clinic as a therapeutic tool. In this article, we reviewed the evolution of experimental culture platforms and introduced a novel iPSCs-based approach for in vitro spermatogenesis based on a niche perspective bearing cellular, chemical, and physical factors that provide the complex arrangement of testicular seminiferous tubules embedded within a vascularized stroma. We believe that bioengineered organoids supported by smart bio-printed tubules and microfluidic organ-on-a-chip systems offer efficient, precise, personalized platforms for autologous pluripotent stem cell sources to undergo the spermatogenetic cycle, presenting a promising tool for infertile male patients with complete testicular aplasia. [ABSTRACT FROM AUTHOR]

Various groups including animal protection organizations, medical organizations, research centers, and even federal agencies such as the U.S. Food and Drug Administration, are working to minimize animal use in scientific experiments. This movement primarily stems from animal welfare and ethical concerns. However, recent advances in technology and new studies in medicine have contributed to an increase in animal experiments throughout the years. With the rapid increase in animal testing, concerns arise including ethical issues, high cost, complex procedures, and potential inaccuracies. Alternative solutions have recently been investigated to address the problems of animal testing. Some of these technologies are related to stem cell technologies, such as organ-on-a-chip, organoids, and induced pluripotent stem cell models. The aim of the review is to focus on stem cell related methodologies, such as organoids, that can serve as an alternative to animal testing and discuss its advantages and limitations, alongside regulatory considerations. Although stem cell related methodologies has shortcomings, it has potential to replace animal testing. Achieving this requires further research on stem cells, with potential societal and technological benefits. [ABSTRACT FROM AUTHOR]

Critical reprogramming factors resided predominantly in the oocyte or male pronucleus can enhance the efficiency or the quality of induced pluripotent stem cells (iPSCs) induction. However, few reprogramming factors exist in the male pronucleus had been verified. Here, we demonstrated that granulin (Grn), a factor enriched specifically in male pronucleus, can significantly improve the generation of iPSCs from mouse fibroblasts. Grn is highly expressed on Day 1, Day 3, Day 14 of reprogramming induced by four Yamanaka factors and functions at the initial stage of reprogramming. Transcriptome analysis indicates that Grn can promote the expression of lysosome‐related genes, while inhibit the expression of genes involved in DNA replication and cell cycle at the early reprogramming stage. Further verification determined that Grn suppressed cell proliferation due to the arrest of cell cycle at G2/M phase. Moreover, ectopic Grn can enhance the lysosomes abundance and rescue the efficiency reduction of reprogramming resulted from lysosomal protease inhibition. Taken together, we conclude that Grn serves as an activator for somatic cell reprogramming through mitigating cell hyperproliferation and promoting the function of lysosomes. [ABSTRACT FROM AUTHOR]

The ideal cell source for articular cartilage repair remains elusive. Using developmentally inspired differentiation protocols, we induced human pluripotent stem cells (hPSCs) toward articular chondrocytes capable of joint cartilage repair in rodent models, which were distinct from growth plate chondrocytes, fated to be replaced by bone in vivo. Working toward clinical translation, we demonstrated controlled differentiation into chondrocytes by comprehensive gene expression analysis at each step of the differentiation. Articular chondrocytes derived from hPSCs could be expanded several passages in vitro without losing chondrogenic potential. Furthermore, chondrocytes isolated from these articular cartilage tissues had the potential to serially regenerate new articular and growth plate cartilage tissues. Finally, the ability to cryopreserve articular chondrocytes with the desired phenotype is critical for clinical translation and here we report no loss in cell viability or regenerative potential following cryopreservation. These results support the immense potential of hPSC‐derived articular chondrocytes as a cell‐based therapy for cartilage repair. [ABSTRACT FROM AUTHOR]

Nitrosative stress is a feature of Alzheimer's disease (AD). Aims: We aimed to identify the cause underpinning increased nitric oxide (NO) in neurons and the impact of NO on neuronal function in AD. Results: We analyzed neuronal nitric oxide synthase (nNOS) protein levels in postmortem tissue and induced pluripotent stem cell (iPSC)-derived neurons from Alzheimer's patients and controls by immunohistochemistry and Western blots. Furthermore, we assessed the impact of modulating nNOS function or NO levels on neuronal glutamatergic signaling using calcium imaging. We show that nNOS protein levels are increased in early and severely affected brain regions of AD postmortem tissue, but not late and mildly affected regions, or cognitively normal individuals. The increased nNOS phenotype was also present in iPSC-derived neurons from late-onset Alzheimer's disease (LOAD) patients compared with controls, along with increased levels of nitrite, a stable marker of NO. Innovation: We observed a divergent functional impact of NO that included strengthening the calcium response in control neurons, while dysregulating calcium signaling and altering the amplitude and kinetics of the calcium responses to glutamate in the AD neurons. Pharmacological scavenging of NO or inhibition of nNOS prevented aberrant spontaneous calcium signaling in AD neurons. Conclusion: Together these data identify increases in nNOS protein in AD. Functional data suggest that NO modulation of glutamatergic calcium signaling is neuroprotective under nonpathogenic conditions, with increased nNOS and NO contributing to dysregulated spontaneous calcium signaling in AD neurons. [ABSTRACT FROM AUTHOR]

Biobanks of human biosamples and cell lines are indispensable for biomedical research on human health and disease and for drug development projects. Many human cell line biobanks worldwide hold collections of lymphoblastoid cell lines (LCLs), representing thousands of affected and control donors from diverse ethnic/ancestry groups. In recent years, induced human pluripotent stem cells (iPSCs) and differentiated human cells derived from these iPSCs have become indispensable for applied biomedical research. Establishing iPSCs remains a laborious and costly step towards generating differentiated human cells. To address this research need, several non‐profit and commercial biobanks have established iPSC collections for distribution to researchers, thereby serving as a resource for generating differentiated human cells. The most common starting materials for generation of iPSCs are a skin biopsy for harvesting fibroblasts, or a blood sample for collection of peripheral blood mononuclear cells. However untapped resources include the large established collections of biobanked human LCLs which can be reprogrammed to iPSCs using a variety of published protocols including the use of non‐integrating episomal vectors. Many biobanks curate LCLs from diverse ethnic/ancestry populations, an aspect largely absent in most established iPSC biobanks which tend to primarily reflect populations from developed countries. Here, we call upon researchers across the breadth of iPSC research to tap the unique resource of existing and diverse human LCL collections for establishing biobanked iPSC panels that better represent the varied human ethnic (and hence genomic) diversity, thereby benefiting precision medicine and drug development research on a global scale. [ABSTRACT FROM AUTHOR]

A mechanism exploration is an important part of toxicological studies. However, traditional cell and animal models can no longer meet the current needs for in-depth studies of toxicological mechanisms. The three-dimensional (3D) organoid derived from human embryonic stem cells (hESC) or induced pluripotent stem cells (hiPSC) is an ideal experimental model for the study of toxicological effects and mechanisms, which further recapitulates the human tissue microenvironment and provides a reliable method for studying complex cell–cell interactions. This article provides a comprehensive overview of the state of the 3D organoid technology in toxicological studies, including a bibliometric analysis of the existing literature and an exploration of the latest advances in toxicological mechanisms. The use of 3D organoids in toxicology research is growing rapidly, with applications in disease modeling, organ-on-chips, and drug toxicity screening being emphasized, but academic communications among countries/regions, institutions, and research scholars need to be further strengthened. Attempts to study the toxicological mechanisms of exogenous chemicals such as heavy metals, nanoparticles, drugs and organic pollutants are also increasing. It can be expected that 3D organoids can be better applied to the safety evaluation of exogenous chemicals by establishing a standardized methodology. [ABSTRACT FROM AUTHOR]

Inflammatory bowel diseases (IBDs) are prevalent and debilitating diseases with limited clinical treatment strategies. Mesenchymal stem cell (MSCs) are pluripotent stem cells with self-renewal capability and multiple immunomodulatory effects, which make them a promising therapeutic approach for IBDs. Thus, optimization of MSCs regimes is crucial for their further clinical application. Wogonin, a flavonoid-like compound with extensive immunomodulatory and adjuvant effects, has been investigated as a potential pretreatment for MSCs in IBD treatment. In this study, we employed the DSS-induced acute colitis mouse model to compare the therapeutic effectiveness of MSCs in pretreated with or without wogonin and further explore the underlying mechanism. Compared to untreated MSCs, MSCwogonin (pretreated with wogonin) showed greater effectiveness in the treatment of colitis. Further experiments revealed that wogonin treatment activated the AKT signaling pathway, resulting in higher cellular glycolysis. Inhibition of AKT phosphorylation by perifosine not only decreased glycolysis but impaired the therapeutic efficiency of MSCwogonin. Consistent with these results, qPCR data indicated that wogonin treatment induced the expression of immunomodulatory molecules IL-10, IDO, and AGR1, which were reduced by perifosine. Together, our data demonstrated that wogonin preconditioning strategy further augmented the therapeutic efficacy of MSCs via promoting glycolysis, which should be a promising strategy for optimizing MSCs therapy in IBDs. [ABSTRACT FROM AUTHOR]

Allogeneic natural killer (NK) cell therapy has been effective in treating cancer. Many studies have tested NK cell therapy using human pluripotent stem cells (hPSCs). However, the impacts of the origin of PSC-NK cells on competence are unclear. In this study, several types of hPSCs, including human-induced PSCs (hiPSCs) generated from CD34+, CD3−CD56+, and CD56− cells in umbilical cord blood (UCB), three lines of human embryonic stem cells (hESCs, ES-1. ES-2 and ES-3) and MHC I knockout (B2M-KO)-ESCs were used to differentiate into NK cells and their capacities were analyzed. All PSC types could differentiate into NK cells. Among the iPSC-derived NK cells (iPSC-NKs) and ESC-derived NK cells (ES-NKs), 34+ iPSCs and ES-3 had a higher growth rate and cytotoxicity, respectively, ES-3 also showed better efficacy than 34+ iPSCs. B2M-KO was similar to the wild type. These results suggest that the screening for differentiation of PSCs into NK cells prior to selecting the PSC lines for the development of NK cell immunotherapy is an essential process for universal allotransplantation, including the chimeric antigen receptor (CAR). [ABSTRACT FROM AUTHOR]

Neutrophils are the most frequent immune cell type in peripheral blood, performing an essential role against pathogens. People with neutrophil deficiencies are susceptible to deadly infections, highlighting the importance of generating these cells in host immunity. Neutrophils can be generated from hematopoietic progenitor cells (HPCs) and embryonic stem cells (ESCs) using a cocktail of cytokines. In addition, induced pluripotent stem cells (iPSCs) can be differentiated into various functional cell types, including neutrophils. iPSCs can be derived from differentiated cells, such as skin and blood cells, by reprogramming them to a pluripotent state. Neutrophil generation from iPSCs involves a multistep process that can be performed through feeder cell-dependent and feeder cell-independent manners. Various cytokines and growth factors, in particular, stem cell facto, IL-3, thrombopoietin and granulocyte colony-stimulating factor (G-CSF), are used in both methods, especially, G-CSF which induces the final differentiation of neutrophils in the granulocyte lineage. iPSC-derived neutrophils have been used as a valuable tool for studying rare genetic disorders affecting neutrophils. The iPSC-derived neutrophils can also be used for disease modeling, infection research and drug discovery. However, several challenges must be overcome before iPSC-derived neutrophils can be used therapeutically in transplantation medicine. This review provides an overview of the commonly employed protocols for generating neutrophils from HPCs, ESCs and iPSCs and discusses the potential applications of the generated cells in research and medicine. [Display omitted] • Neutrophils have been successfully differentiated from hematopoietic progenitor cells and iPSCs. • Neutrophil generation from iPSCs has been done via feeder cell-dependent and independent manners. • iPSC-derived neutrophils have been used for studying various neutrophil genetic disorders. • iPSC-derived neutrophils can be used for effective adoptive cellular therapy, drug development and disease modeling. [ABSTRACT FROM AUTHOR]

Stem cells have gained attention as a promising therapeutic approach for damaged myocardium, and there have been efforts to develop a protocol for regenerating cardiomyocytes (CMs). Certain cells have showed a greater aptitude for yielding beating CMs, such as induced pluripotent stem cells, embryonic stem cells, adipose-derived stromal vascular fraction cells and extended pluripotent stem cells. The approach for generating CMs from stem cells differs across studies, although there is evidence that Wnt signaling, chemical additives, electrical stimulation, co-culture, biomaterials and transcription factors triggers CM differentiation. Upregulation of Gata4, Mef2c and Tbx5 transcription factors has been correlated with successfully induced CMs, although Mef2c may potentially play a more prominent role in the generation of the beating phenotype, specifically. Regenerative research provides a possible candidate for cardiac repair; however, it is important to identify factors that influence their differentiation. Altogether, the spontaneously beating CMs would be monumental for regenerative research for cardiac repair. [ABSTRACT FROM AUTHOR]

Metabolic dysfunction-associated Fatty Liver Disease (MAFLD) has emerged as one of the leading cardiometabolic diseases. Friend of GATA2 (FOG2) is a transcriptional co-regulator that has been shown to regulate hepatic lipid metabolism and accumulation. Using meta-analysis from several different biobank datasets, we identified a coding variant of FOG2 (rs28374544, A1969G, S657G) predominantly found in individuals of African ancestry (minor allele frequency~20%), which is associated with liver failure/cirrhosis phenotype and liver injury. To gain insight into potential pathways associated with this variant, we interrogated a previously published genomics dataset of 38 human induced pluripotent stem cell (iPSCs) lines differentiated into hepatocytes (iHeps). Using Differential Gene Expression Analysis and Gene Set Enrichment Analysis, we identified the mTORC1 pathway as differentially regulated between iHeps from individuals with and without the variant. Transient lipid-based transfections were performed on the human hepatoma cell line (Huh7) using wild-type FOG2 and FOG2S657G and demonstrated that FOG2S657G increased mTORC1 signaling, de novo lipogenesis, and cellular triglyceride synthesis and mass. In addition, we observed a significant downregulation of oxidative phosphorylation in FOG2S657G cells in fatty acid-loaded cells but not untreated cells, suggesting that FOG2S657G may also reduce fatty acid to promote lipid accumulation. Taken together, our multi-pronged approach suggests a model whereby the FOG2S657G may promote MAFLD through mTORC1 activation, increased de novo lipogenesis, and lipid accumulation. Our results provide insights into the molecular mechanisms by which FOG2S657G may affect the complex molecular landscape underlying MAFLD. [ABSTRACT FROM AUTHOR]

Coronary microvascular disease (CMD) and its progression towards major adverse coronary events pose a significant health challenge. Accurate in vitro investigation of CMD requires a robust cell model that faithfully represents the cells within the cardiac microvasculature. Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) offer great potential; however, they are traditionally derived via differentiation protocols that are not readily scalable and are not specified towards the microvasculature. Here, we report the development and comprehensive characterisation of a scalable 3D protocol enabling the generation of phenotypically stable cardiac hPSC-microvascular-like ECs (hPSC-CMVECs) and cardiac pericyte-like cells. These were derived by growing vascular organoids within 3D stirred tank bioreactors and subjecting the emerging 3D hPSC-ECs to high-concentration VEGF-A treatment (3DV). Not only did this promote phenotypic stability of the 3DV hPSC-ECs; single cell-RNA sequencing (scRNA-seq) revealed the pronounced expression of cardiac endothelial- and microvascular-associated genes. Further, the generated mural cells attained from the vascular organoid exhibited markers characteristic of cardiac pericytes. Thus, we present a suitable cell model for investigating the cardiac microvasculature as well as the endothelial-dependent and -independent mechanisms of CMD. Moreover, owing to their phenotypic stability, cardiac specificity, and high angiogenic potential, the cells described within would also be well suited for cardiac tissue engineering applications. [ABSTRACT FROM AUTHOR]

Background: B-cell CLL/lymphoma 6 member B (BCL6B) operates as a sequence-specific transcriptional repressor within the nucleus, playing crucial roles in various biological functions, including tumor suppression, immune response, stem cell self-renew, and vascular angiogenesis. However, whether BCL6B is involved in endothelial cell (EC) development has remained largely unknown. ETS variant transcription factor 2 (ETV2) is well known to facilitate EC differentiation. This study aims to determine the important role of BCL6B in EC differentiation and its potential mechanisms. Methods: Doxycycline-inducible human induced pluripotent stem cell (hiPSC) lines with BCL6B overexpression or BCL6B knockdown were established and subjected to differentiate into ECs and vessel organoids (VOs). RNA sequencing analysis was performed to identify potential signal pathways regulated by BCL6B during EC differentiation from hiPSCs. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of pluripotency and vascular-specific marker genes expression. EC differentiation efficiency was determined by Flow cytometry analysis. The performance of EC was evaluated by in vitro Tube formation assay. The protein expression and the vessel-like structures were assessed using immunofluorescence analysis or western blot. Luciferase reporter gene assay and chromatin immunoprecipitation (ChIP)-PCR analysis were used to determine the regulatory relationship between BCL6B and ETV2. Results: Functional ECs and VOs were successfully generated from hiPSCs. Notably, overexpression of BCL6B suppressed while knockdown of BCL6B improved EC differentiation from hiPSCs. Additionally, the overexpression of BCL6B attenuated the capacity of derived hiPSC-ECs to form a tubular structure. Furthermore, compared to the control VOs, BCL6B overexpression repressed the growth of VOs, whereas BCL6B knockdown had little effect on the size of VOs. RNA sequencing analysis confirmed that our differentiation protocol induced landscape changes for cell/tissue/system developmental process, particularly vascular development and tube morphogenesis, which were significantly modulated by BCL6B. Subsequent experiments confirmed the inhibitory effect of BCL6B is facilitated by the binding of BCL6B to the promoter region of ETV2, led to the suppression of ETV2's transcriptional activity. Importantly, the inhibitory effect of BCL6B overexpression on EC differentiation from hiPSCs could be rescued by ETV2 overexpression. Conclusions: BCL6B inhibits EC differentiation and hinders VO development by repressing the transcriptional activity of ETV2. [ABSTRACT FROM AUTHOR]

Background: While pluripotent stem cell (PSC) therapies move toward clinical and commercial applications at a rapid rate, manufacturing reproducibility and robustness are notable bottlenecks in regulatory approval. Therapeutic applications of PSCs require large cell quantities to be generated under highly robust, well-defined, and economically viable conditions. Small-scale and short-term process optimization, however, is often performed in a linear fashion that does not account for time needed to verify the bioprocess protocols and analysis methods used. Design of a reproducible and robust bioprocess should be dynamic and include a continuous effort to understand how the process will respond over time and to different stresses before transitioning into large-scale production where stresses will be amplified. Methods: This study utilizes a baseline protocol, developed for the short-term culture of PSC aggregates in Vertical-Wheel® bioreactors, to evaluate key process attributes through long-term (serial passage) suspension culture. This was done to access overall process robustness when performed with various commercially available media and cell lines. Process output variables including growth kinetics, aggregate morphology, harvest efficiency, genomic stability, and functional pluripotency were assessed through short and long-term culture. Results: The robust nature of the expansion protocol was demonstrated over a six-day culture period where spherical aggregate formation and expansion were observed with high-fold expansions for all five commercial media tested. Profound differences in cell growth and quality were revealed only through long-term serial expansion and in-vessel dissociation operations. Some commercial media formulations tested demonstrated maintenance of cell growth rates, aggregate morphology, and high harvest recovery efficiencies through three bioreactor serial passages using multiple PSC lines. Exceptional bioprocess robustness was even demonstrated with sustained growth and quality maintenance over 10 serial bioreactor passages. However, some commercial media tested proved less equipped for serial passage cultures in bioreactors as cultures led to cell lysis during dissociation, reduction in growth rates, and a loss of aggregate morphology. Conclusions: This study demonstrates the importance of systematic selection and testing of bioprocess input variables, with multiple bioprocess output variables through serial passages to create a truly reproducible and robust protocol for clinical and commercial PSC production using scalable bioreactor systems. [ABSTRACT FROM AUTHOR]

Nerve regeneration and circuit reconstruction remain a challenge following spinal cord injury (SCI). Corticospinal pyramidal neurons possess strong axon projection ability. In this study, human induced pluripotent stem cells (iPSCs) were differentiated into pyramidal neuronal precursors (PNPs) by addition of small molecule dorsomorphin into the culture. iPSC-derived PNPs were transplanted acutely into a rat contusion SCI model on the same day of injury. Following engraftment, the SCI rats showed significantly improved motor functions compared with vehicle control group as revealed by behavioral tests. Eight weeks following engraftment, the PNPs matured into corticospinal pyramidal neurons and extended axons into distant host spinal cord tissues, mostly in a caudal direction. Host neurons rostral to the lesion site also grew axons into the graft. Possible synaptic connections as a bridging relay may have been formed between host and graft-derived neurons, as indicated by pre- and post-synaptic marker staining and the regulation of chemogenetic regulatory systems. PNP graft showed an anti-inflammatory effect at the injury site and could bias microglia/macrophages towards a M2 phenotype. In addition, PNP graft was safe and no tumor formation was detected after transplantation into immunodeficient mice and SCI rats. The potential to reconstruct a neuronal relay circuitry across the lesion site and to modulate the microenvironment in SCI makes PNPs a promising cellular candidate for treatment of SCI. [ABSTRACT FROM AUTHOR]

Today, human pluripotent stem cell technologies find widespread application across biomedical research, as models for early human development, as platforms for functional human genomics, as tools for the study of disease, drug screening and toxicology, and as a renewable source of cellular therapeutics for a range of intractable diseases. The foundations of this human pluripotent stem cell revolution rest on advances in a wide range of disciplines, including cancer biology, assisted reproduction, cell culture and organoid technology, somatic cell nuclear transfer, primate embryology, single‐cell biology, and gene editing. This review surveys the slow emergence of the study of human pluripotency and the exponential growth of the field during the past several decades. [ABSTRACT FROM AUTHOR]

Dysfunction of pancreatic δ cells contributes to the etiology of diabetes. Despite their important role, human δ cells are scarce, limiting physiological studies and drug discovery targeting δ cells. To date, no directed δ-cell differentiation method has been established. Here, we demonstrate that fibroblast growth factor (FGF) 7 promotes pancreatic endoderm/progenitor differentiation, whereas FGF2 biases cells towards the pancreatic δ-cell lineage via FGF receptor 1. We develop a differentiation method to generate δ cells from human stem cells by combining FGF2 with FGF7, which synergistically directs pancreatic lineage differentiation and modulates the expression of transcription factors and SST activators during endoderm/endocrine precursor induction. These δ cells display mature RNA profiles and fine secretory granules, secrete somatostatin in response to various stimuli, and suppress insulin secretion from in vitro co-cultured β cells and mouse β cells upon transplantation. The generation of human pancreatic δ cells from stem cells in vitro would provide an unprecedented cell source for drug discovery and cell transplantation studies in diabetes. Human pancreatic δ cells play important roles in the balance of pancreatic hormones. Here, the authors develop a directed differentiation method to generate in vitro and in vivo functional δ cells from human stem cells by combining FGF2 with FGF7. [ABSTRACT FROM AUTHOR]

Stem cell-based therapies have emerged as a promising approach for treating various neurological disorders by harnessing the regenerative potential of stem cells to restore damaged neural tissue and circuitry. This comprehensive review provides an in-depth analysis of the current state of stem cell applications in primary neurological conditions, including Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), stroke, spinal cord injury (SCI), and other related disorders. The review begins with a detailed introduction to stem cell biology, discussing the types, sources, and mechanisms of action of stem cells in neurological therapies. It then critically examines the preclinical evidence from animal models and early human trials investigating the safety, feasibility, and efficacy of different stem cell types, such as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), and induced pluripotent stem cells (iPSCs). While ESCs have been studied extensively in preclinical models, clinical trials have primarily focused on adult stem cells such as MSCs and NSCs, as well as iPSCs and their derivatives. We critically assess the current state of research for each cell type, highlighting their potential applications and limitations in different neurological conditions. The review synthesizes key findings from recent, high-quality studies for each neurological condition, discussing cell manufacturing, delivery methods, and therapeutic outcomes. While the potential of stem cells to replace lost neurons and directly reconstruct neural circuits is highlighted, the review emphasizes the critical role of paracrine and immunomodulatory mechanisms in mediating the therapeutic effects of stem cells in most neurological disorders. The article also explores the challenges and limitations associated with translating stem cell therapies into clinical practice, including issues related to cell sourcing, scalability, safety, and regulatory considerations. Furthermore, it discusses future directions and opportunities for advancing stem cell-based treatments, such as gene editing, biomaterials, personalized iPSC-derived therapies, and novel delivery strategies. The review concludes by emphasizing the transformative potential of stem cell therapies in revolutionizing the treatment of neurological disorders while acknowledging the need for rigorous clinical trials, standardized protocols, and multidisciplinary collaboration to realize their full therapeutic promise. [ABSTRACT FROM AUTHOR]

In 2013, M. Lancaster described the first protocol to obtain human brain organoids. These organoids, usually generated from human-induced pluripotent stem cells, can mimic the three-dimensional structure of the human brain. While they recapitulate the salient developmental stages of the human brain, their use to investigate the onset and mechanisms of neurodegenerative diseases still faces crucial limitations. In this review, we aim to highlight these limitations, which hinder brain organoids from becoming reliable models to study neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Specifically, we will describe structural and biological impediments, including the lack of an aging footprint, angiogenesis, myelination, and the inclusion of functional and immunocompetent microglia--all important factors in the onset of neurodegeneration in AD, PD, and ALS. Additionally, we will discuss technical limitations for monitoring the microanatomy and electrophysiology of these organoids. In parallel, we will propose solutions to overcome the current limitations, thereby making human brain organoids a more reliable tool to model neurodegeneration. [ABSTRACT FROM AUTHOR]

Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC–derived cholangiocytes expressed markers of mature cholangiocytes, such as CK7, CK19, and cystic fibrosis transmembrane conductance regulator (CFTR). Compared with the 2D culture system–generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes YAP1 and JAG1 and extrahepatic cholangiocytes AQP1 and MUC1. Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and CFTR channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)- mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies. [ABSTRACT FROM AUTHOR]

Background: Commonly used media for the differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CMs) contain high concentrations of proteins, in particular albumin, which is prone to quality variations and presents a substantial cost factor, hampering the clinical translation of in vitro-generated cardiomyocytes for heart repair. To overcome these limitations, we have developed chemically defined, entirely protein-free media based on RPMI, supplemented with L-ascorbic acid 2-phosphate (AA-2P) and either the non-ionic surfactant Pluronic F-68 or a specific polyvinyl alcohol (PVA). Methods and Results: Both media compositions enable the efficient, directed differentiation of embryonic and induced hPSCs, matching the cell yields and cardiomyocyte purity ranging from 85 to 99% achieved with the widely used protein-based CDM3 medium. The protein-free differentiation approach was readily up-scaled to a 2000 mL process scale in a fully controlled stirred tank bioreactor in suspension culture, producing > 1.3 × 109 cardiomyocytes in a single process run. Transcriptome analysis, flow cytometry, electrophysiology, and contractile force measurements revealed that the mass-produced cardiomyocytes differentiated in protein-free medium exhibit the expected ventricular-like properties equivalent to the well-established characteristics of CDM3-control cells. Conclusions: This study promotes the robustness and upscaling of the cardiomyogenic differentiation process, substantially reduces media costs, and provides an important step toward the clinical translation of hPSC-CMs for heart regeneration. [ABSTRACT FROM AUTHOR]

Aims Understanding the molecular identity of human pluripotent stem cell (hPSC)-derived cardiac progenitors and mechanisms controlling their proliferation and differentiation is valuable for developmental biology and regenerative medicine. Methods and results Here, we show that chemical modulation of histone acetyl transferases (by IQ-1) and WNT (by CHIR99021) synergistically enables the transient and reversible block of directed cardiac differentiation progression on hPSCs. The resulting stabilized cardiovascular progenitors (SCPs) are characterized by ISL1pos/KI-67pos/NKX2-5neg expression. In the presence of the chemical inhibitors, SCPs maintain a proliferation quiescent state. Upon small molecules, removal SCPs resume proliferation and concomitant NKX2-5 up-regulation triggers cell-autonomous differentiation into cardiomyocytes. Directed differentiation of SCPs into the endothelial and smooth muscle lineages confirms their full developmental potential typical of bona fide cardiovascular progenitors. Single-cell RNA-sequencing-based transcriptional profiling of our in vitro generated human SCPs notably reflects the dynamic cellular composition of E8.25-E9.25 posterior second heart field of mouse hearts, hallmarked by nuclear receptor sub-family 2 group F member 2 expression. Investigating molecular mechanisms of SCP stabilization, we found that the cell-autonomously regulated retinoic acid and BMP signalling is governing SCP transition from quiescence towards proliferation and cell-autonomous differentiation, reminiscent of a niche-like behaviour. Conclusion The chemically defined and reversible nature of our stabilization approach provides an unprecedented opportunity to dissect mechanisms of cardiovascular progenitors' specification and reveal their cellular and molecular properties. [ABSTRACT FROM AUTHOR]

Naïve epiblast cells in the embryo and pluripotent stem cells in vitro undergo developmental progression to a formative state competent for lineage specification. During this transition, transcription factors and chromatin are rewired to encode new functional features. Here, we examine the role of mitogen-activated protein kinase (ERK1/2) signalling in pluripotent state transition. We show that a primary consequence of ERK activation in mouse embryonic stem cells is elimination of Nanog, which precipitates breakdown of the naïve state gene regulatory network. Variability in pERK dynamics results in heterogeneous loss of Nanog and metachronous state transition. Knockdown of Nanog allows exit without ERK activation. However, transition to formative pluripotency does not proceed and cells collapse to an indeterminate identity. This outcome is due to failure to maintain expression of the central pluripotency factor Oct4. Thus, during formative transition ERK signalling both dismantles the naïve state and preserves pluripotency. These results illustrate howa single signalling pathway can both initiate and secure transition between cell states. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) still poses a challenge in terms of complex etiology, not fully effective methods of treatment, and lack of healing agents. This neurodegenerative condition considerably affects the comfort of life by causing difficulties with movement and worsening cognition. Neuron, astrocyte, microglia, and oligodendrocyte activity is engaged in multiple pathogenic processes associated with MS. These cells are also utilized in creating in vitro cellular models for investigations focusing on MS. In this article, we present and discuss a summary of different in vitro models useful for MS research and describe their development. We discuss cellular models derived from animals or humans and present in the form of primary cell lines or immortalized cell lines. In addition, we characterize cell cultures developed from induced pluripotent stem cells (iPSCs). Culture conditions (2D and 3D cultures) are also discussed. [ABSTRACT FROM AUTHOR]

The apolipoprotein E4 (APOE4) allele represents the major genetic risk factor for Alzheimer's disease (AD). In contrast, APOE2 is known to lower the AD risk, while APOE3 is defined as risk neutral. APOE plays a prominent role in the bioenergetic homeostasis of the brain, and early-stage metabolic changes have been detected in the brains of AD patients. Although APOE is primarily expressed by astrocytes in the brain, neurons have also been shown as source for APOE. However, the distinct roles of the three APOE isoforms in neuronal energy homeostasis remain poorly understood. In this study, we generated pure human neurons (iN cells) from APOE-isogenic induced pluripotent stem cells (iPSCs), expressing either APOE2, APOE3, APOE4, or carrying an APOE knockout (KO) to investigate APOE isoform-specific effects on neuronal energy metabolism. We showed that endogenously produced APOE4 enhanced mitochondrial ATP production in APOE-isogenic iN cells but not in the corresponding iPS cell line. This effect neither correlated with the expression levels of mitochondrial fission or fusion proteins nor with the intracellular or secreted levels of APOE, which were similar for APOE2, APOE3, and APOE4 iN cells. ATP production and basal respiration in APOE-KO iN cells strongly differed from APOE4 and more closely resembled APOE2 and APOE3 iN cells, indicating a gain-of-function mechanism of APOE4 rather than a loss-of-function. Taken together, our findings in APOE isogenic iN cells reveal an APOE genotype-dependent and neuron-specific regulation of oxidative energy metabolism. [ABSTRACT FROM AUTHOR]

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP. [ABSTRACT FROM AUTHOR]

Genetic or hereditary kidney disease stands as a pivotal cause of chronic kidney disease (CKD). The proliferation and widespread utilization of DNA testing in clinical settings have notably eased the diagnosis of genetic kidney diseases, which were once elusive but are now increasingly identified in cases previously deemed CKD of unknown etiology. However, despite these diagnostic strides, research into disease pathogenesis and novel drug development faces significant hurdles, chiefly due to the dearth of appropriate animal models and the challenges posed by limited patient cohorts in clinical studies. Conversely, the advent and utilization of human-induced pluripotent stem cells (hiPSCs) offer a promising avenue for genetic kidney disease research. Particularly, the development of hiPSC-derived kidney organoid systems presents a novel platform for investigating various forms of genetic kidney diseases. Moreover, the integration of the CRISPR/Cas9 technique into this system holds immense potential for efficient research on genetic kidney diseases. This review aims to explore the applications of in vitro kidney organoids generated from hiPSCs in the study of diverse genetic kidney diseases. Additionally, it will delve into the limitations of this research platform and outline future perspectives for advancing research in this crucial area. [ABSTRACT FROM AUTHOR]

Autism spectrum disorders (ASDs) are complex neurodevelopmental conditions characterized by deficits in social interaction and communication, as well as repetitive behaviors. Although the etiology of ASD is multifactorial, with both genetic and environmental factors contributing to its development, a strong genetic basis is widely recognized. Recent research has identified numerous genetic mutations and genomic rearrangements associated with ASD-characterizing genes involved in brain development. Alterations in developmental programs are particularly harmful during critical periods of brain development. Notably, studies have indicated that genetic disruptions occurring during the second trimester of pregnancy affect cortical development, while disturbances in the perinatal and early postnatal period affect cerebellar development. The developmental defects must be viewed in the context of the role of the cerebellum in cognitive processes, which is now well established. The present review emphasizes the genetic complexity and neuropathological mechanisms underlying ASD and aims to provide insights into the cerebellar involvement in the disorder, focusing on recent advances in the molecular landscape governing its development in humans. Furthermore, we highlight when and in which cerebellar neurons the ASD-associated genes may play a role in the development of cortico–cerebellar circuits. Finally, we discuss improvements in protocols for generating cerebellar organoids to recapitulate the long period of development and maturation of this organ. These models, if generated from patient-induced pluripotent stem cells (iPSC), could provide a valuable approach to elucidate the contribution of defective genes to ASD pathology and inform diagnostic and therapeutic strategies. [ABSTRACT FROM AUTHOR]

To protect against the constant threat of inhaled pathogens, the lung is equipped with cellular defenders. In coordination with resident and recruited immune cells, this defence is initiated by the airway and alveolar epithelium following their infection with respiratory viruses. Further support for viral clearance and infection resolution is provided by adjacent endothelial and stromal cells. However, even with these defence mechanisms, respiratory viral infections are a significant global health concern, causing substantial morbidity, socioeconomic losses, and mortality, underlining the need to develop effective vaccines and antiviral medications. In turn, the identification of new treatment options for respiratory infections is critically dependent on the availability of tractable in vitro experimental models that faithfully recapitulate key aspects of lung physiology. For such models to be informative, it is important these models incorporate human-derived, physiologically relevant versions of all cell types that normally form part of the lungs anti-viral response. This review proposes a guideline using human induced pluripotent stem cells (iPSCs) to create all the disease-relevant cell types. iPSCs can be differentiated into lung epithelium, innate immune cells, endothelial cells, and fibroblasts at a large scale, recapitulating in vivo functions and providing genetic tractability. We advocate for building comprehensive iPSC-derived in vitro models of both proximal and distal lung regions to better understand and model respiratory infections, including interactions with chronic lung diseases. [ABSTRACT FROM AUTHOR]

Studying human fetal lungs can inform how developmental defects and disease states alter the function of the lungs. Here, we sequenced >150,000 single cells from 19 healthy human pseudoglandular fetal lung tissues ranging between gestational weeks 10–19. We capture dynamic developmental trajectories from progenitor cells that express abundant levels of the cystic fibrosis conductance transmembrane regulator (CFTR). These cells give rise to multiple specialized epithelial cell types. Combined with spatial transcriptomics, we show temporal regulation of key signalling pathways that may drive the temporal and spatial emergence of specialized epithelial cells including ciliated and pulmonary neuroendocrine cells. Finally, we show that human pluripotent stem cell-derived fetal lung models contain CFTR-expressing progenitor cells that capture similar lineage developmental trajectories as identified in the native tissue. Overall, this study provides a comprehensive single-cell atlas of the developing human lung, outlining the temporal and spatial complexities of cell lineage development and benchmarks fetal lung cultures from human pluripotent stem cell differentiations to similar developmental window. Quach and Farrell et al. report single-cell transcriptomic analysis of over 150,000 cell from 19 human fetal lung tissues and describe the temporal and spatial dynamics of epithelial lineage development. These epithelial lineage trajectories were further identified in human pluripotent stem cell-based models of lung cell differentiation. [ABSTRACT FROM AUTHOR]