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Bats are tolerant to highly pathogenic viruses such as Marburg, Ebola, and Nipah, suggesting the presence of a unique immune tolerance toward viral infection. Here, we compared severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of human and bat (Rhinolophus ferrumequinum) pluripotent cells and fibroblasts. Since bat cells do not express an angiotensin-converting enzyme 2 (ACE2) receptor that allows virus infection, we transduced the human ACE2 (hA) receptor into the cells and found that transduced cells can be infected with SARS-CoV-2. Compared to human embryonic stem cells-hA, infected bat induced Pluripotent Stem Cells (iPSCs)-hA produced about a 100-fold lower level of infectious virus and displayed lower toxicity. In contrast, bat embryonic fibroblast-hA produced no infectious virus while being infectable and synthesizing viral RNA and proteins, suggesting abortive infection. Indeed, electron microscopy failed to detect virus-like particles in infected bat fibroblasts in contrast to bat iPSCs or human cells, consistent with the latter producing infectious viruses. This suggests that bat somatic but not pluripotent cells have an effective mechanism to control virus replication. Consistent with previous results by others, we find that bat cells have a constitutively activated innate immune system, which might limit SARS-CoV-2 infection compared to human cells. [ABSTRACT FROM AUTHOR]

Alport syndrome (AS) is a hereditary disease caused by mutations in the COL4A5 gene and leads to chronic kidney disease. Currently, no specific treatment has been developed. However, a recent study using AS‐model mice demonstrated that the exon skipping method could partially rescue the symptoms. In this study, we evaluated the effects of the exon skipping method using kidney organoids generated from AS‐patient‐derived induced pluripotent stem cells (AS‐iPSCs). We generated kidney organoids from AS‐iPSCs, which exhibited nephron structures. As expected, the C‐terminus of COL4A5 was not expressed in AS‐organoids. Interestingly, anti‐sense oligonucleotides restored the expression of the C‐terminus of COL4A5 in vitro. Next, we transplanted AS‐organoids into mice and evaluated glomerular basement membrane formation in vivo. We found that AS‐organoids formed a lower slit diaphragm ratio compared to control organoids. Finally, we assessed the effects of exon skipping on transplanted organoids but observed minimum effects. These studies suggest that AS‐iPSCs can generate kidney organoids lacking the C‐terminus of COL4A5, and that exon skipping can induce its expression in vitro. [ABSTRACT FROM AUTHOR]

Purpose: The ability to generate natural killer (NK) cells from induced pluripotent stem cells (iPSCs) has given rise to new possibilities for the large-scale production of homogeneous immunotherapeutic cellular products and opened new avenues towards the creation of "off-the-shelf" cancer immunotherapies. However, the differentiation of NK cells from iPSCs remains poorly understood, particularly regarding the ontogenic landscape of iPSC-derived NK (iNK) cells produced in vitro and the influence that the differentiation strategy employed may have on the iNK profile. Methods: To investigate this question, we conducted a comparative analysis of two sets of iNK cells generated from the same iPSC line using two different protocols: (i) a short-term, clinically compatible feeder-free protocol corresponding to primitive hematopoiesis, and (ii) a lymphoid-based protocol representing the definitive hematopoietic step. Results and discussion: Our work demonstrated that both protocols are capable of producing functional iNK cells. However, the two sets of resulting iNKs exhibited distinct phenotypes and transcriptomic profiles. The lymphoid-based differentiation approach generated iNKs with a more mature and activated profile, which demonstrated higher cytotoxicity against cancer cell lines compared to iNK cells produced under short-term feeder-free conditions suggesting that the differentiation strategy must be considered when designing iNK cell-based adoptive immunotherapies. [ABSTRACT FROM AUTHOR]

Stem cell therapies for Parkinson's disease are at an exciting time of development, and several clinical trials have recently been initiated. Human pluripotent stem cells are differentiated into transplantable dopamine (DA) progenitors which are proliferative at the time of grafting and undergo terminal differentiation and maturation in vivo. While the progenitors are homogeneous at the time of transplantation, they give rise to heterogeneous grafts composed not only of therapeutic DA neurons but also of other mature cell types. The mechanisms for graft diversification are unclear. We used single-nucleus RNA-seq and ATAC-seq to profile DA progenitors before transplantation combined with molecular barcode-based tracing to determine origin and shared lineages of the mature cell types in the grafts. Our data demonstrate that astrocytes, vascular leptomeningeal cells, and DA neurons are the main component of the DAergic grafts, originating from a common progenitor that is tripotent at the time of transplantation. [ABSTRACT FROM AUTHOR]

The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) has led to numerous advances in the field of retinal research, with great potential for the use of hPSC-derived RGCs for studies of human retinal development, in vitro disease modeling, drug discovery, as well as their potential use for cell replacement therapeutics. Of all these possibilities, the use of hPSC-derived RGCs as a human-relevant platform for in vitro disease modeling has received the greatest attention, due to the translational relevance as well as the immediacy with which results may be obtained compared to more complex applications like cell replacement. While several studies to date have focused upon the use of hPSC-derived RGCs with genetic variants associated with glaucoma or other optic neuropathies, many of these have largely described cellular phenotypes with only limited advancement into exploring dysfunctional cellular pathways as a consequence of the disease-associated gene variants. Thus, to further advance this field of research, in the current study we leveraged an isogenic hPSC model with a glaucoma-associated mutation in the Optineurin (OPTN) protein, which plays a prominent role in autophagy. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor AMPK, along with subsequent neurodegeneration in OPTN(E50K) RGCs differentiated from hPSCs, and have further validated some of these findings in a mouse model of ocular hypertension. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs, while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN(E50K) RGCs. Taken together, these results highlighted that autophagy disruption resulted in increased autophagic demand which was associated with downregulated signaling through mTORC1, contributing to the degeneration of RGCs. [ABSTRACT FROM AUTHOR]

Systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, immune dysregulation, and systemic fibrosis. Research on SSc has been hindered largely by lack of relevant models to study the progressive nature of the disease and to recapitulate the cell plasticity that is observed in this disease context. Generation of models for fibrotic disease using pluripotent stem cells is important for recapitulating the heterogeneity of the fibrotic tissue and are a potential platform for screening anti-fibrotic drugs. We previously reported a novel in-vitro model for fibrosis using induced pluripotent stem cell-derived mesenchymal cells (iSCAR). Here we report the generation of a "scar-like phenotype" when iPSC derived mesenchymal cells are cultured on hydrogel that mimicks a wound healing/scarring response (iSCAR). First, we performed RNA sequencing (RNA-seq) based transcriptome profiling of iSCAR culture at 48 h and 13 days to characterize early and latestage scarring phenotypes. The next generation RNA-seq of these iSCAR culture at different timepoints detected expression 92% of early "scar associated" genes and 85% late "scar associated" genes, respectively. Comparative transcriptomic analysis of a gene level SSc compendium matrix to the iSCAR wound associated model revealed genes common in both data sets. Early scar formation genes showed biological processes of hypoxia (27.5%), vascular development (13.7%) and glycolysis (27.5), while late scar formation showed genes associated with senescence (22.6%). Next we show the effects of two different antifibrotic compounds to validate the utility of the model as a screening tool to study early and late-stagelate-stage fibrosis. An autotaxin inhibitor was used to validate the iSCAR late stage fibrotic model (iSCAR-T) and an antifibrotic tool screening compound of unknown mechanism (EX00015097) was used to study and validate both early (iSCAR-P) and late-stage (iSCAR-T) fibrosis in the iSCAR model. [ABSTRACT FROM AUTHOR]

Puerarin, a natural isoflavone, is commonly used as a Chinese herbal medicine for the treatment of various cardiovascular and neurological disorders. It has been found to be neuroprotective via TrK‐PI3K/Akt pathway, which is associated with anti‐inflammatory and antioxidant effects. Myelin damage in diseases such as multiple sclerosis (MS) and ischemia induces activation of endogenous oligodendrocyte progenitor cells (OPC) and subsequent remyelination by newly formed oligodendrocytes. It has been shown that human‐induced pluripotent stem cells (hiPSC)‐derived OPCs promote remyelination when transplanted to the brains of disease models. Here, we ask whether and how puerarin is beneficial to the generation of hiPSC‐derived OPCs and oligodendrocytes, and to the endogenous remyelination in mouse demyelination model. Our results show that puerarin increases the proportion of O4+ pre‐oligodendrocytes differentiated from iPSC‐derived neural stem cells. In vitro, puerarin increases proliferation of rat OPCs and enhances mitochondrial activity. Treatment of puerarin at progenitor stage increases the yielding of differentiated oligodendrocytes. In rat organotypic brain slice culture, puerarin promotes both myelination and remyelination. In vivo, puerarin increases oligodendrocyte repopulation during remyelination in mouse spinal cord following lysolethicin‐induced demyelination. Our findings suggest that puerarin promotes oligodendrocyte lineage progression and myelin repair, with a potential to be developed into therapeutic agent for neurological diseases associated with myelin damage. [ABSTRACT FROM AUTHOR]

The intricate network of protein-chaperone interactions is crucial for maintaining cellular function. Recent discoveries have unveiled the existence of specialized chaperone assemblies, known as epichaperomes, which serve as scaffolding platforms that orchestrate the reconfiguration of protein-protein interaction networks, thereby enhancing cellular adaptability and proliferation. This study explores the structural and regulatory aspects of epichaperomes, with a particular focus on the role of post-translational modifications (PTMs) in their formation and function. A key finding is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 within an intrinsically disordered region, as critical determinants of epichaperome assembly. Our data demonstrate that phosphorylation of these serine residues enhances HSP90's interactions with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Moreover, we establish a direct link between epichaperome function and cellular physiology, particularly in contexts where robust proliferation and adaptive behavior are essential, such as in cancer and pluripotent stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone assemblies in diseases characterized by epichaperome dysregulation, thereby bridging the gap between fundamental research and precision medicine. Epichaperomes are chaperone assemblies that reorganize protein networks under stress. Here, authors reveal how HSP90 phosphorylation drives epichaperome formation, linking this process to cancer cell behaviors and pluripotent stem cell functions. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function. Pluripotent cells are defined by their ability to self-renew: to proliferate indefinitely while maintaining their identity. Here, authors use genome-scale CRISPR knockout screens to untangle these two aspects of self-renewal. [ABSTRACT FROM AUTHOR]

Introduction: Reprogramming of human-induced pluripotent stem cells (iPSCs) and their differentiation into specific cell types, such as induced sensory-like neurons (iSNs), are critical for disease modeling and drug testing. However, the variability of cell populations challenges reliability and reproducibility. While various protocols for iSN differentiation exist, the development of non-iSN cells in these cultures remains an issue. Therefore, standardization of protocols is essential. This study aimed to improve iSN culture conditions by reducing the number of non-iSN cells while preserving the survival and quality of iSNs. Methods: iSNs were differentiated from a healthy control iPSC line using an established protocol. Interventions for protocol optimization included floxuridine (FdU) or 1-b-D-arabinofuranosyl-cytosine hydrochloride (AraC) treatment, magnetic-activated cell sorting (MACS), early cell passaging, and replating. Cell viability and iSN-to-total-cell-count ratio were assessed using a luminescent assay and immunocytochemistry, respectively. Results: Passaging of cells during differentiation did not increase the iSN-to- total-cell-count ratio, and MACS of immature iSNs led to neuronal blebbing and reduced the iSN-to-total-cell-count ratio. Treatment with high concentrations and prolonged incubation of FdU or AraC resulted in excessive cell death. However, treatment with 10µM FdU for 24 h post-differentiation showed the most selective targeting of non-iSN cells, leading to an increase in the iSN-to-total-cell count ratio without compromising the viability or functionality of the iSN population. Replating of iSNs shortly after seeding also helped to reduce non-iSN cells. Conclusion: In direct comparison with other methods, treatment with 10µM FdU for 24 h after differentiation shows promise for improving iSN culture purity, which could benefit downstream applications in disease modeling and drug discovery. However, further investigations involving multiple iPSC lines and optimization of protocol parameters are warranted to fully exploit the potential of thismethod and enhance its reproducibility and applicability.Overall, this study provides valuable insights into optimizing culture conditions for iSN differentiation and highlights the importance of standardized protocols in iPSC- based research. [ABSTRACT FROM AUTHOR]

Inherited retinal diseases (IRDs) represent a diverse group of genetic disorders leading to progressive degeneration of the retina due to mutations in over 280 genes. This review focuses on the various methodologies for the preclinical characterization and evaluation of adeno-associated virus (AAV)-mediated gene therapy as a potential treatment option for IRDs, particularly focusing on gene therapies targeting mutations, such as those in the RPE65 and FAM161A genes. AAV vectors, such as AAV2 and AAV5, have been utilized to deliver therapeutic genes, showing promise in preserving vision and enhancing photoreceptor function in animal models. Despite their advantages—including high production efficiency, low pathogenicity, and minimal immunogenicity—AAV-mediated therapies face limitations such as immune responses beyond the retina, vector size constraints, and challenges in large-scale manufacturing. This review systematically compares different experimental models used to investigate AAV-mediated therapies, such as mouse models, human retinal explants (HREs), and induced pluripotent stem cell (iPSC)-derived retinal organoids. Mouse models are advantageous for genetic manipulation and detailed investigations of disease mechanisms; however, anatomical differences between mice and humans may limit the translational applicability of results. HREs offer valuable insights into human retinal pathophysiology but face challenges such as tissue degradation and lack of systemic physiological effects. Retinal organoids, on the other hand, provide a robust platform that closely mimics human retinal development, thereby enabling more comprehensive studies on disease mechanisms and therapeutic strategies, including AAV-based interventions. Specific outcomes targeted in these studies include vision preservation and functional improvements of retinas damaged by genetic mutations. This review highlights the strengths and weaknesses of each experimental model and advocates for their combined use in developing targeted gene therapies for IRDs. As research advances, optimizing AAV vector design and delivery methods will be critical for enhancing therapeutic efficacy and improving clinical outcomes for patients with IRDs. [ABSTRACT FROM AUTHOR]

Aging represents the leading risk factor for developing neurodegenerative disorders. One of the nine hallmarks of aging is mitochondrial dysfunction. Age-related mitochondrial alterations have been shown to affect mitochondrial energy metabolism, reduction-oxidation homeostasis, and mitochondrial dynamics. Previous reports have shown that induced pluripotent stem cells (iPSCs) from aged donors do not keep the aging signature at the transcriptomic level. However, not all aspects of aging have been investigated, and especially not the mitochondria-related aging signature. Therefore, the present study compared the mitochondrial function in iPSCs from healthy aged donors compared to those of young donors. We addressed whether aged iPSCs may be used as drug-screening models of "aging in a dish" to identify therapies alleviating mitochondria aging. Compared to iPSCs from young donors, we demonstrate that iPSCs from aged donors show impaired mitochondrial bioenergetics and exhibit a rise in reactive oxygen species generation. Furthermore, aged iPSCs present a lower mitochondrial mass and alterations in the morphology of the mitochondrial network when compared to iPSCs from young donors. This study provides the first evidence that the aging phenotype is present at the mitochondrial level in iPSCs from aged donors, ranging from bioenergetics to mitochondrial network morphology. This model might be used to screen mitochondria-targeting drugs to promote healthy aging at the mitochondrial level. [ABSTRACT FROM AUTHOR]

Stem cell therapy constitutes a pivotal subject in contemporary discourse, with donor stem cells having been employed in research and clinical treatments for several decades. Primary cell transplantation encompasses diverse stem cell types, including ectomesenchymal stem cells, hematopoietic stem cells, and various stem cell derivatives such as vesicles and extracellular vesicles. Nevertheless, the emergence of cell engineering techniques has heralded a new epoch in stem cell therapy, markedly broadening their therapeutic potential. Induced pluripotent stem cells (iPSCs) epitomize a significant milestone in modern medical biology. This groundbreaking discovery offers significant potential in disciplines such as biology, pathophysiology, and cellular regenerative medicine. As a result, iPSCs derived differentiated cells have become a pioneering avenue for cell therapy research. Induced mesenchymal stem cells (iMSCs), derived from iPSCs, represent a novel frontier in MSCs related research. Empirical evidence suggests that iMSCs demonstrate enhanced proliferative capacities compared to natural MSCs, with diminished age-related variability and heterogeneity. Numerous clinical trials have highlighted the prospective superiority of iMSCs. This article synthesizes current basic research and clinical trials pertaining to iMSCs, aiming to provide a reference point for future research endeavors. [ABSTRACT FROM AUTHOR]

Soluble biochemical agents are being employed to generate kidney organoids from human‐induced pluripotent stem cells. However, this soluble factor approach does not consider the effect of the mechanical environment on lineage commitment. Here, a mechanoresponsive nano‐environment with cell‐adhesive properties composed of supramolecular hydrogelators that co‐assemble into fibrous superstructures to form a transient network is presented, which is used to encapsulate kidney organoids. The delayed sol‐gel transition of the transient network enables fibrous superstructures to diffuse into the densely packed extracellular organoid space during the encapsulation procedure. This allows the mechanoresponsive matrix to induce a biological response beyond the organoid‐hydrogel border, and tune glomerulogenesis inside kidney organoids. In this manner, biomaterials are used as a complementary tool to soluble biochemical agents in tuning lineage commitment and refining organoid maturation. [ABSTRACT FROM AUTHOR]

Pluripotent stem cell lines derived from preimplantation mouse embryos have opened opportunities for the study of early mammalian development and generation of genetically uncompromised material for differentiation into specific cell types. Murine embryonic stem cells are highly versatile and can be engineered and introduced into host embryos, transferred to recipient females, and gestated to investigate gene function at multiple levels as well as developmental mechanisms, including lineage segregation and cell competition. In this review, we summarize the biomedical motivation driving the incremental modification to culture regimes and analyses that have advanced stem cell research to its current state. Ongoing investigation into divergent mechanisms of early developmental processes adopted by other species, such as agriculturally beneficial mammals and birds, will continue to enrich knowledge and inform strategies for future in vitro models. [ABSTRACT FROM AUTHOR]

Nerve-derived factors have attracted attention in bone regeneration therapy due to their ability to promote bone regeneration and nerve innervation. Mesenchymal stem cells transported to target sites promote osteogenesis. However, there are few reports on the effects of neural stem cells on bone regeneration. Therefore, the aim of this study was to investigate the role of neural stem cells in osteogenesis. Here, embryoid bodies (EB) or primary neurospheres (1NS) were generated using mouse induced pluripotent stem cells (iPS cells), which were then seeded onto gelatin (Gel) sponges. The seeded Gel sponges were then transplanted into mouse calvarial bone defects. We noted that 1NS-seeded Gel promoted bone regeneration and the presence of tartrate-resistant acid phosphatase (TRAP)-positive cells, whereas the EB-seeded Gel did not. RNA-sequencing of the 1NS-seeded and EB seeded Gels showed an upregulation of the transforming growth factor (TGF)-β signaling pathway in the 1NS-seeded Gel group. Immunostaining confirmed the presence of Id3 positive cells in mice with bone defects treated with the 1NS-seeded Gel. These findings suggest that the transplantation of neural stem cells may contribute to the promotion of bone regeneration. This study aimed to investigate whether neural stem cells, when seeded in Gel sponges, promoted bone regeneration. It has been well documented that bone is tightly linked with the nervous systems. Bioscaffolds comprising factors that promote innervation and bone regeneration have been investigated for use in bone therapy. However, there is limited research on the use of neural stem cells for promoting bone formation. To assess this relationship, we conducted both in vivo and in vitro assays to determine whether neural stem cells promoted bone formation. We noted that 1NS-seeded Gel sponges promoted bone formation significantly in mice with calvarial defects after 4 weeks. This study provides a novel approach of neural stem cells for bone therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Low Earth Orbit (LEO) has emerged as a unique environment for evaluating altered stem cell properties in microgravity. LEO has become increasingly accessible for research and development due to progress in private spaceflight. Axiom Mission 2 (Ax-2) was launched as the second all-private astronaut mission to the International Space Station (ISS). Frozen human induced pluripotent stem cells (hiPSCs) expressing green fluorescent protein (GFP) under the SOX2 promoter, as well as fibroblasts differentiated from SOX2-GFP hiPSCs, were sent to the ISS. Astronauts then thawed and seeded both cell types into commercially available 96-well plates, which provided surface tension that reduced fluid movement out of individual wells and showed that hiPSCs or hiPSC-derived fibroblasts could survive either in suspension or attached to a Matrigel substrate. Furthermore, both cell types could be transfected with red fluorescent protein (RFP)-expressing plasmid. We demonstrate that hiPSCs and hiPSC-fibroblasts can be thawed in microgravity in off-the-shelf, commercially-available cell culture hardware, can associate into 3D spheroids or grow adherently in Matrigel, and can be transfected with DNA. This lays the groundwork for future biomanufacturing experiments in space. [ABSTRACT FROM AUTHOR]

Background: Three common isoforms of the apolipoprotein E (APOE) gene - APOE2, APOE3, and APOE4 - hold varying significance in Alzheimer's Disease (AD) risk. The APOE4 allele is the strongest known genetic risk factor for late-onset Alzheimer's Disease (AD), and its expression has been shown to correlate with increased central nervous system (CNS) amyloid deposition and accelerated neurodegeneration. Conversely, APOE2 is associated with reduced AD risk and lower CNS amyloid burden. Recent clinical data have suggested that increased blood-brain barrier (BBB) leakage is commonly observed among AD patients and APOE4 carriers. However, it remains unclear how different APOE isoforms may impact AD-related pathologies at the BBB. Methods: To explore potential impacts of APOE genotypes on BBB properties and BBB interactions with amyloid beta, we differentiated isogenic human induced pluripotent stem cell (iPSC) lines with different APOE genotypes into both brain microvascular endothelial cell-like cells (BMEC-like cells) and brain pericyte-like cells. We then compared the effect of different APOE isoforms on BBB-related and AD-related phenotypes. Statistical significance was determined via ANOVA with Tukey's post hoc testing as appropriate. Results: Isogenic BMEC-like cells with different APOE genotypes had similar trans-endothelial electrical resistance, tight junction integrity and efflux transporter gene expression. However, recombinant APOE4 protein significantly impeded the "brain-to-blood" amyloid beta 1–40 (Aβ40) transport capabilities of BMEC-like cells, suggesting a role in diminished amyloid clearance. Conversely, APOE2 increased amyloid beta 1–42 (Aβ42) transport in the model. Furthermore, we demonstrated that APOE-mediated amyloid transport by BMEC-like cells is dependent on LRP1 and p-glycoprotein pathways, mirroring in vivo findings. Pericyte-like cells exhibited similar APOE secretion levels across genotypes, yet APOE4 pericyte-like cells showed heightened extracellular amyloid deposition, while APOE2 pericyte-like cells displayed the least amyloid deposition, an observation in line with vascular pathologies in AD patients. Conclusions: While APOE genotype did not directly impact general BMEC or pericyte properties, APOE4 exacerbated amyloid clearance and deposition at the model BBB. Conversely, APOE2 demonstrated a potentially protective role by increasing amyloid transport and decreasing deposition. Our findings highlight that iPSC-derived BBB models can potentially capture amyloid pathologies at the BBB, motivating further development of such in vitro models in AD modeling and drug development. [ABSTRACT FROM AUTHOR]

Acute coronary syndromes, such as myocardial infarction (MI), lack effective therapies beyond heart transplantation, which is often hindered by donor scarcity and postoperative complications. Human induced pluripotent stem cells (hiPSCs) offer the possibility of myocardial regeneration by differentiating into cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-cardiomyocytes) exhibit fetal-like calcium flux and energy metabolism, which inhibits their engraftment. Several strategies have been explored to improve the therapeutic efficacy of hiPSC-cardiomyocytes, such as selectively enhancing energy substrate utilization and improving the transplantation environment. In this review, we have discussed the impact of altered mitochondrial biogenesis and metabolic switching on the maturation of hiPSC-cardiomyocytes. Additionally, we have discussed the limitations inherent in current methodologies for assessing metabolism in hiPSC-cardiomyocytes, and the challenges in achieving sufficient metabolic flexibility akin to that in the healthy adult heart. [ABSTRACT FROM AUTHOR]

The establishment of epiblast-derived pluripotent stem cells (PSCs) from cattle, which are important domestic animals that provide humans with milk and meat while also serving as bioreactors for producing valuable proteins, poses a challenge due to the unclear molecular signaling required for embryonic epiblast development and maintenance of PSC self-renewal. Here, we selected six key stages of bovine embryo development (E5, E6, E7, E10, E12, and E14) to track changes in pluripotency and the dependence on signaling pathways via modified single-cell transcription sequencing technology. The remarkable similarity of the gene expression patterns between cattle and pigs during embryonic lineage development contributed to the successful establishment of bovine epiblast stem cells (bEpiSCs) using 3i/LAF (WNTi, GSK3βi, SRCi, LIF, Activin A, and FGF2) culture system. The generated bEpiSCs exhibited consistent expression patterns of formative epiblast pluripotency genes and maintained clonal morphology, normal karyotypes, and proliferative capacity for more than 112 passages. Moreover, these cells exhibited high-efficiency teratoma formation as well as the ability to differentiate into various cell lineages. The potential of bEpiSCs for myogenic differentiation, primordial germ cell like cells (PGCLCs) induction, and as donor cells for cell nuclear transfer was also assessed, indicating their promise in advancing cell-cultured meat production, gene editing, and animal breeding. [ABSTRACT FROM AUTHOR]

Fibronectin (FN) is an extracellular matrix glycoprotein essential for the development and function of major vertebrate organ systems. Mutations in FN result in an autosomal dominant skeletal dysplasia termed corner fracture-type spondylometaphyseal dysplasia (SMDCF). The precise pathomechanisms through which mutant FN induces impaired skeletal development remain elusive. Here, we have generated patient-derived induced pluripotent stem cells as a cell culture model for SMDCF to investigate the consequences of FN mutations on mesenchymal stem cells (MSCs) and their differentiation into cartilage-producing chondrocytes. In line with our previous data, FN mutations disrupted protein secretion from MSCs, causing a notable increase in intracellular FN and a significant decrease in extracellular FN levels. Analyses of plasma samples from SMDCF patients also showed reduced FN in circulation. FN and endoplasmic reticulum (ER) protein folding chaperones (BIP, HSP47) accumulated in MSCs within ribosome-covered cytosolic vesicles that emerged from the ER. Massive amounts of these vesicles were not cleared from the cytosol, and a smaller subset showed the presence of lysosomal markers. The accumulation of intracellular FN and ER proteins elevated cellular stress markers and altered mitochondrial structure. Bulk RNA sequencing revealed a specific transcriptomic dysregulation of the patient-derived cells relative to controls. Analysis of MSC differentiation into chondrocytes showed impaired mesenchymal condensation, reduced chondrogenic markers, and compromised cell proliferation in mutant cells. Moreover, FN mutant cells exhibited significantly lower transforming growth factor beta-1 (TGFβ1) expression, crucial for mesenchymal condensation. Exogenous FN or TGFβ1 supplementation effectively improved the MSC condensation and promoted chondrogenesis in FN mutant cells. These findings demonstrate the cellular consequences of FN mutations in SMDCF and explain the molecular pathways involved in the associated altered chondrogenesis. Significance/Highlights: • SMDCF-causing mutations in fibronectin impair protein secretion in iPSC-derived mesenchymal stem cells. • Mutant fibronectin and ER protein folding chaperones are directly exported from the rough endoplasmic reticulum into vesicles covered with ribosomes, some of which are positive for lysosomal markers. • The FN mutant cells are characterized by massive accumulation of cytosolic vesicles. • Mutations in fibronectin impair stem cell proliferation, mesenchymal condensation, and the differentiation of MSCs into chondrocytes. • Exogenous addition of purified fibronectin or TGFβ-1 improves mesenchymal condensation and chondrogenesis of the FN mutant stem cells. [ABSTRACT FROM AUTHOR]

Neuroblastoma is the most common pediatric extracranial solid tumor and is derived from trunk neural crest cells (tNCC) and its progenitor sympathoadrenal (SA) cells. While human pluripotent stem cell (PSC) models of neuroblastoma have been described, the PSC were differentiated using protocols that made neural crest cells, but not specifically the trunk subtype. Here, we compared four recent protocols to differentiate pluripotent stem cells (PSC) toward SA cells and examined their efficiency at generating SA cells along with earlier cell states (neuromesodermal progenitors [NMP], tNCC), as well as generating MYCN-driven tumors. Interestingly, the protocols that created cells with the highest level of NMP markers did not produce cells with the highest tNCC or SA cell markers. We identified a protocol that consistently produced cells with the highest level of SA markers using two PSC lines of different genders. This protocol also generated tumors with the highest level of PHOX2B, a marker of neuroblastoma. Transcriptionally, however, each protocol generates tumors that resemble neuroblastoma. Two of the protocols repeatedly produced adrenergic neuroblastoma whereas the other two protocols were ambiguous. Thus, we identified a protocol that reliably generates adrenergic neuroblastoma. [ABSTRACT FROM AUTHOR]

The application of organoids derived from animal tissues and human-induced pluripotent stem cells to safety assessments of environmental chemicals has been introduced over the last decade. One of the objectives of this approach is to develop an alternative method for animal toxicological studies, while another is to focus on the local reactions of chemicals in each organ/tissue. One of the most important goals is bridging the toxicological properties of chemicals between animals and humans, which may be compared on a level playing field using healthy organoids derived from both animals and humans in vitro, excluding species difference in the absorption, distribution, metabolism, and excretion properties of chemicals in vivo. An overview of the application of organoid systems to safety assessments of environmental chemicals, including general toxicology, developmental toxicology, carcinogenicity, and mutagenicity, was provided herein, and bridging strategies using both animal and human organoids are proposed as a future perspective. [ABSTRACT FROM AUTHOR]

Stem cell fate decisions, including proliferation, differentiation, morphological changes, and viability, are impacted by microenvironmental cues such as physical and biochemical signals. However, the specific impact of matrix elasticity on kidney cell development and function remains less understood due to the lack of models that can closely recapitulate human kidney biology. An established protocol to differentiate podocytes from human-induced pluripotent stem (iPS) cells provides a promising avenue to elucidate the role of matrix elasticity in kidney tissue development and lineage determination. In this study, we synthesized polyacrylamide hydrogels with different stiffnesses and investigated their ability to promote podocyte differentiation and biomolecular characteristics. We found that 3 kPa and 10 kPa hydrogels significantly support the adhesion, differentiation, and viability of podocytes. Differentiating podocytes on a more compliant (0.7 kPa) hydrogel resulted in significant cell loss and detachment. Further investigation of the mechanosensitive proteins yes-associated protein (YAP) and synaptopodin revealed nuanced molecular distinctions in cellular responses to matrix elasticity that may otherwise be overlooked if morphology and cell spreading alone were used as the primary metric for selecting matrices for podocyte differentiation. Specifically, hydrogels with kidney-like rigidities outperformed traditional tissue culture plates at modulating the molecular-level expression of active mechanosensitive proteins critical for podocyte health and function. These findings could guide the development of physiologically relevant platforms for kidney tissue engineering, disease modeling, and mechanistic studies of organ physiology and pathophysiology. Such advances are critical for realizing the full potential of in vitro platforms in accurately predicting human biological responses. [ABSTRACT FROM AUTHOR]

Background/Objectives: Age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are leading causes of vision loss, with AMD affecting older populations and RP being a rarer, genetically inherited condition. Both diseases result in progressive retinal degeneration, for which current treatments remain inadequate in advanced stages. This review aims to provide an overview of the retina's anatomy and physiology, elucidate the pathophysiology of AMD and RP, and evaluate emerging cell-based therapies for these conditions. Methods: A comprehensive review of the literature was conducted, focusing on cell therapy approaches, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and retinal progenitor cells. Preclinical and clinical studies were analyzed to assess therapeutic potential, with attention to mechanisms such as cell replacement, neuroprotection, and paracrine effects. Relevant challenges, including ethical concerns and clinical translation, were also explored. Results: Cell-based therapies demonstrate potential for restoring retinal function and slowing disease progression through mechanisms like neuroprotection and cell replacement. Preclinical trials show promising outcomes, but clinical studies face significant hurdles, including challenges in cell delivery and long-term efficacy. Combination therapies integrating gene editing and biomaterials offer potential future advancements. Conclusions: While cell-based therapies for AMD and RP have made significant progress, substantial barriers to clinical application remain. Further research is essential to overcome these obstacles, improve delivery methods, and ensure the safe and effective translation of these therapies into clinical practice. [ABSTRACT FROM AUTHOR]

Basal cells are adult stem cells in the airway epithelium and regenerate differentiated cell populations, including the mucosecretory and ciliated cells that enact mucociliary clearance. Human basal cells can proliferate and produce differentiated epithelium in vitro. However, studies of airway epithelial differentiation mostly rely on immunohistochemical or immunofluorescence-based staining approaches, meaning that a dynamic approach is lacking, and quantitative data are limited. Here, we use a lentiviral reporter gene approach to transduce primary human basal cells with bioluminescence reporter constructs to monitor airway epithelial differentiation longitudinally. We generated three constructs driven by promoter sequences from the TP63, MUC5AC, and FOXJ1 genes to quantitatively assess basal cell, mucosecretory cell, and ciliated cell abundance, respectively. We validated these constructs by tracking differentiation of basal cells in air-liquid interface and organoid ("bronchosphere") cultures. Transduced cells also responded appropriately to stimulation with interleukin 13 (IL-13; to increase mucosecretory differentiation and mucus production) and IL-6 (to increase ciliated cell differentiation). These constructs represent a new tool for monitoring airway epithelial cell differentiation in primary epithelial and/or induced pluripotent stem cell (iPSC)-derived cell cultures. NEW & NOTEWORTHY: Orr et al. generated and validated new lentiviral vectors to monitor the differentiation of airway basal cells, goblet cells, or multiciliated cells using bioluminescence. [ABSTRACT FROM AUTHOR]

The objective of the current study is to develop a new method for tracking transplanted human induced pluripotent stem cells‐derived cardiomyocytes (hiPSC‐CMs) using magnetic resonance imaging (MRI). The CRISPR/dCas9 activation system is employed to overexpress ferritin heavy chain (FHC) in hiPSC‐CMs. The mRNA and protein expression of FHC in hiPSC and hiPSC‐CMs significantly increased after transfection. Iron chloride does not affect the cell viability in a concentration range from 0 to 2000 µm. hiPSCs overexpressing FHC (hiPSC‐ FHCOE) and hiPSC‐CMs overexpressing FHC (hiPSC‐CM‐FHCOE) significantly enhanced cellular uptake of iron chloride but with no changes in electrophysiological properties compared to hiPSC‐CM‐Control. Furthermore, hiPSC‐CM‐FHCOE presented robust contrast and lower T2* values, signifying their potential as highly effective candidates for cardiac MRI. Next, hiPSC‐CM‐FHCOE is injected into mouse hearts and after 3 days of transplantation, MR images are obtained. hiPSC‐CM‐FHCOE cells exhibited clear signals in the hearts with lower T2* and rapid signal decay. Collectively, data from this proof‐of‐concept study demonstrated that endogenous labeling with FHC in hiPSC‐CMs can be a potent strategy for enhancing the accuracy of cardiac MRI. This technology represents a significant step forward in tracking the transplanted hiPSC‐CMs in the hearts of live animals. [ABSTRACT FROM AUTHOR]

Cellular plasticity progressively declines with development and differentiation, yet these processes can be experimentally reversed by reprogramming somatic cells to induced pluripotent stem cells (iPSCs) using defined transcription factors. Advances in reprogramming technology over the past 15 years have enabled researchers to study diseases with patient-specific iPSCs, gain fundamental insights into how cell identity is maintained, recapitulate early stages of embryogenesis using various embryo models, and reverse aspects of aging in cultured cells and animals. Here, we review and compare currently available reprogramming approaches, including transcription factor-based methods and small molecule-based approaches, to derive pluripotent cells characteristic of early embryos. Additionally, we discuss our current understanding of mechanisms that resist reprogramming and their role in cell identity maintenance. Finally, we review recent efforts to rejuvenate cells and tissues with reprogramming factors, as well as the application of iPSCs in deriving novel embryo models to study pre-implantation development. [ABSTRACT FROM AUTHOR]

Micropatterning is reliable method for quantifying pluripotency of human‐induced pluripotent stem cells (hiPSCs) that differentiate to form a spatial pattern of sorted, ordered and nonoverlapped three germ layers on the micropattern. In this study, we propose a deep learning method to quantify spatial patterning of the germ layers in the early differentiation stage of hiPSCs using micropattern images. We propose decoding and encoding U‐net structures learning labelled Hoechst (DNA‐stained) hiPSC regions with corresponding Hoechst and bright‐field micropattern images to segment hiPSCs on Hoechst or bright‐field images. We also propose a U‐net structure to extract extraembryonic regions on a micropattern, and an algorithm to compares intensities of the fluorescence images staining respective germ‐layer cells and extract their regions. The proposed method thus can quantify the pluripotency of a hiPSC line with spatial patterning including cell numbers, areas and distributions of germ‐layer and extraembryonic cells on a micropattern, and reveal the formation process of hiPSCs and germ layers in the early differentiation stage by segmenting live‐cell bright‐field images. In our assay, the cell‐number accuracy achieved 86% and 85%, and the cell region accuracy 89% and 81% for segmenting Hoechst and bright‐field micropattern images, respectively. Applications to micropattern images of multiple hiPSC lines, micropattern sizes, groups of markers, living and fixed cells show the proposed method can be expected to be a useful protocol and tool to quantify pluripotency of a new hiPSC line before providing it to the scientific community. LAY DESCRIPTION: Human‐induced pluripotent stem cells (hiPSCs) are an ideal resource for patient specific regenerative medicine. Pluripotency of a hiPSC line has to be analysed before us. Differentiated hiPSCs on a micropattern have been used for the analysis on which hiPSCs form segregated, sorted and radially ordered three germ layers. This study uses DNA‐stained fluorescence microscopy images for segmentation of the differentiated hiPSCs, together with fluorescence images staining respective ectoderm, mesoderm and endoderm to classify a segmented hiPSC as any of the three germ layers. However, cells on a DNA‐stained fluorescence micropattern image are low‐contrast to background and dense that are not easily segmented by image‐processing methods. Meanwhile, regions of fluorescent pixels on the germ‐layer fluorescence images overlap, although the hiPSCs differentiating on a micropattern expand in a two‐dimensional, nonoverlapped way at the early differentiation stage. We propose a U‐net structure with flexible input micropattern sizes and with designed loss functions to identify the dense differentiated hiPSCs to improve segmentation accuracy. We then use an intensity comparison calculation that compares fluorescence intensities of all the corresponding pixels on the germ‐layer fluorescence images inside a segmented hiPSC to classify the cell into a nonoverlapped region of any germ layer. Then, distribution (spatial pattering) of a germ layer on the micropattern are quantified using numbers, areas and distances to the micropattern centre of cells inside the germ layer. The U‐net is also trained by the labelled cells on DNA‐stained fluorescence images with their corresponding bright‐field images. This enables the hiPSC segmentation on live‐cell time‐lapse bright‐field micropattern images to quantify spatial patterning changes of the germ layers over time or monitoring of their formation process. [ABSTRACT FROM AUTHOR]

Regenerative medicine shows significant potential in treating kidney diseases through the application of various types of stem and progenitor cells, including mesenchymal stem cells (MSCs), renal stem/progenitor cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Stem cells possess the unique ability to repair injured organs and improve impaired functions, making them a key element in the research of therapies for kidney tissue repair and organ regeneration. In kidney transplantation, reperfusion injury can cause tissue destruction, leading to an initially low glomerular filtration rate and long-term impact on function by creating irreversible interstitial fibrosis. MSCs have proven useful in repairing early tissue injury in animal models of kidney, lung, heart, and intestine transplantation. The use of stem cell therapies in solid organ transplantation raises the question of whether autologous or allogeneic cells should be preferred. Adipose-derived stem cells (ASCs), characterized by the lack of HLA Class II molecules and low expression of HLA Class I and co-stimulatory signals, are considered immune-privileged. However, the actual risk of graft rejection associated with allogeneic ASCs remains unclear. It has been demonstrated that donor-derived ASCs can promote the development of Treg cells in vitro, and some degree of tolerance induction has been observed in vivo. Nevertheless, a study comparing the efficacy of autologous and allogeneic ASCs in a rat model with a total MHC mismatch for kidney transplantation showed that donor-derived administration of ASCs did not improve the grafts' survival and was associated with increased mortality through an immunologically mediated mechanism. Given the lack of data, autologous ASCs appear to be a safer option in this research context. The aim of this review was to examine the differences between autologous and allogeneic ASCs in the context of their application in kidney transplantation therapies, considering potential immune reactions and therapeutic efficacy. Some have argued that ASCs harvested from end-stage renal disease (ESRD) patients may have lower regenerative potential due to the toxic effects of uremia, potentially limiting their use in transplantation settings. However, evidence suggests that the beneficial properties of ASCs are not affected by uremia or dialysis. Indeed, some investigators have demonstrated that ASCs harvested from chronic kidney disease (CKD) patients exhibit normal characteristics and function, maintaining consistent proliferative capacity and genetic stability over time, even after prolonged exposure to uremic serum Furthermore, no differences were observed in the response of ASCs to immune activation or their inhibitory effect on the proliferation of alloantigen-activated peripheral blood mononuclear cells between patients with normal or impaired renal function. This review presents the current achievements in stem cell research aimed at treating kidney diseases, highlighting significant progress and ongoing efforts in the development of stem cell-based therapies. Despite the encouraging results, further research is needed to overcome the current limitations and fully realize the potential of these innovative treatments. Advances in this field are crucial for developing effective therapies that can address the complex challenges associated with kidney damage and failure. [ABSTRACT FROM AUTHOR]

Human-induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) represent a promising and renewable cell source for therapeutic applications. A systematic evaluation of the immunological properties and engraftment potential of iMSCs generated from urine-derived iPSCs is lacking, which has impeded their broader application. In this study, we differentiated urine-derived iPSCs into iMSCs and assessed their fundamental MSC characteristics, immunogenicity, immunomodulatory capacity and in vivo engraftment. Compared to umbilical cord-derived MSCs (UCMSCs), iMSCs demonstrated an enhanced proliferative capacity, a higher level of regenerative gene expression, and lower immunogenicity, strengthening resistance to apoptosis induced by allogeneic peripheral blood mononuclear cells (PBMCs) and the NK-92 cell line. In addition, iMSCs exhibited a diminished ability to inhibit T cell proliferation and activation compared with UCMSCs. Transcriptomic analyses further revealed the decreased expression of immune regulatory factors in iMSCs. After transfusion into mouse models, iMSCs engrafted in the lungs, liver, and spleen and exhibited the ability to migrate to tumor tissues. Our results indicated that iMSCs generated from urine-derived iPSCs have a significant replicative capacity, low immunogenicity and unique immunomodulatory properties, and hence offer obvious advantages in immune privilege and allogenic therapeutic application prospects. [ABSTRACT FROM AUTHOR]

During a heart attack, ischemia causes losses of billions of cells; this is especially concerning given the minimal regenerative capability of cardiomyocytes (CMs). Heart remuscularization utilizing stem cells has improved cardiac outcomes despite little cell engraftment, thereby shifting focus to cell-free therapies. Consequently, we chose induced pluripotent stem cells (iPSCs) given their pluripotent nature, efficacy in previous studies, and easy obtainability from minimally invasive techniques. Nonetheless, using iPSC secretome-based therapies for treating injured CMs in a clinical setting is ill-understood. We hypothesized that the iPSC secretome, regardless of donor health, would improve cardiovascular outcomes in the CM model of ischemia–reperfusion (IR) injury. Episomal-generated iPSCs from healthy and dilated cardiomyopathy (DCM) donors, passaged 6–10 times, underwent 24 h incubation in serum-free media. Protein content of the secretome was analyzed by mass spectroscopy and used to treat AC16 immortalized CMs during 5 h reperfusion following 24 h of hypoxia. IPSC-derived secretome content, independent of donor health status, had elevated expression of proteins involved in cell survival pathways. In IR conditions, iPSC-derived secretome increased cell survival as measured by metabolic activity (p < 0.05), cell viability (p < 0.001), and maladaptive cellular remodelling (p = 0.052). Healthy donor-derived secretome contained increased expression of proteins related to calcium contractility compared to DCM donors. Congruently, only healthy donor-derived secretomes improved CM intracellular calcium concentrations (p < 0.01). Heretofore, secretome studies mainly investigated differences relating to cell type rather than donor health. Our work suggests that healthy donors provide more efficacious iPSC-derived secretome compared to DCM donors in the context of IR injury in human CMs. These findings illustrate that the regenerative potential of the iPSC secretome varies due to donor-specific differences. [ABSTRACT FROM AUTHOR]

Various age-related disorders accumulate during aging, causing a decline in tissue and organ function, raising the risk of disease development, and leading to death. Age-related phenotypes are tightly related to an increase in coordinated, progressive changes in the transcriptome, proteome, metabolome, microbiome, and epigenome. Age-dependent modifications of the transcriptome, caused by changes in epigenetic, transcriptional, and post-transcriptional regulation of gene expression, lead to the accumulation of age-related changes in the proteome and metabolome. In turn, dynamic changes in the microbiota during aging also affect gene expression and thus lead to age-related changes in the proteome and metabolome. Recent studies have shown that multi-omic rejuvenation technologies decrease age-related disorders and extend longevity. For example, the short-term induction of the expression of transcription factors that ensure the reprogramming of somatic cells into pluripotent stem cells is accompanied by the restoration of the DNA methylation pattern and transcriptome expression profile characteristic of younger tissues, resulting in an increased lifespan. In this review, we discuss existing multi-omic rejuvenation technologies and the prospects for extending and improving life. [ABSTRACT FROM AUTHOR]

In vitro T‐cell differentiation from pluripotent stem cells (PSCs) could potentially provide an unlimited source of T cells for cancer immunotherapy, which, however is still hindered by the inefficient obtaining functionally‐matured, terminally‐differentiated T cells. Here, we established a fluorescence reporter human induced pluripotent stem cell (iPSC) line termed TCF7mCherryRUNX1GFP, in which the endogenous expression of RUNX1 and TCF7 are illustrated by the GFP and mCherry fluorescence, respectively. Utilizing TCF7mCherryRUNX1GFP, we defined that the feeder cells incorporating CXCL12‐expressing OP9 cells with DL4‐expressing OP9 cells at a 1:3 ratio (OP9‐C1D3) significantly enhanced efficiency of CD8+ T cell differentiation from PSCs. Additionally, we engineered a chimeric antigen receptor (CAR) targeting EGFR into iPSCs. The CAR‐T cells differentiated from these iPSCs using OP9‐C1D3 feeders demonstrated effective cytotoxicity toward lung cancer cells. We anticipate this platform will help the in vitro HSPC and T cell differentiation optimization, serving the clinical demands of these cells. [ABSTRACT FROM AUTHOR]

Shwachman–Diamond syndrome (SDS) is an inherited bone marrow failure disorder that often presents at infancy. Progress has been made in revealing causal mutated genes (SBDS and others), ribosome defects, and hematopoietic aberrations in SDS. However, the mechanism underlying the hematopoietic failure remained unknown, and treatment options are limited. Herein, we investigated the onset of SDS embryonic hematopoietic impairments. We generated SDS and control human‐derived induced pluripotent stem cells (iPSCs). SDS iPSCs recapitulated the SDS hematological phenotype. Detailed stepwise evaluation of definitive hematopoiesis revealed defects that started at the early emerging hematopoietic progenitor (EHP) stage after mesoderm and hemogenic endothelium were normally induced. Hematopoietic potential of EHPs was markedly reduced, and the introduction of SBDS in SDS iPSCs improved colony formation. Transcriptome analysis revealed reduced expression of ribosome and oxidative phosphorylation‐related genes in undifferentiated and differentiated iPSCs. However, certain pathways (e.g., DNA replication) and genes (e.g., CHCHD2) were exclusively or more severely dysregulated in EHPs compared with earlier and later stages. To our knowledge, this study offers for the first time an insight into the embryonic onset of human hematopoietic defects in an inherited bone marrow failure syndrome and reveals cellular and molecular aberrations at critical stages of hematopoietic development toward EHPs. [ABSTRACT FROM AUTHOR]

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy causing progressive vision loss due to mutations in the CHM gene, leading to Rab escort protein 1 loss of function. CHM disease is characterized by a progressive degeneration of the choroid, the retinal pigment epithelium (RPE), and the retina. The RPE is a monolayer of polarized cells that supports photoreceptors, providing nutrients, growth factors, and ions, and removes retinal metabolism waste products, having a central role in CHM pathogenesis. Commonly used models such as ARPE-19 cells do not reproduce accurately the nature of RPE cells. Human induced pluripotent stem cells (hiPSCs) can be differentiated into RPE cells (hiPSC-RPE), which mimic key features of native RPE, being more suited to study retinal diseases. Therefore, we took advantage of hiPSCs to generate new human-based CHM models. Two isogenic hiPSC lines were generated through CRISPR/Cas9: a CHM knock-out line from a healthy donor and a corrected CHM patient line using a knock-in approach. The differentiated hiPSC-RPE lines exhibited critical morphological and physiological characteristics of native RPE, including the presence of the tight junction markers Claudin-19 and Zonula Occludens-1, phagocytosis of photoreceptor outer segments, pigmentation, a postmitotic state, and the characteristic polygonal shape. In addition, all the studied cells were able to form retinal organoids. This work resulted in the establishment of isogenic hiPSC lines, representing a new and important CHM cellular model. To our knowledge, this is the first time that isogenic cell lines have been developed to model CHM disease, providing a valuable tool for studying the mechanisms at the onset of RPE degeneration. [ABSTRACT FROM AUTHOR]

Additional sex combs-like 1 (ASXL1) is an epigenetic modulator frequently mutated in myeloid malignancies, generally associated with poor prognosis. Current models for ASXL1-mutated diseases are mainly based on the complete deletion of Asxl1 or overexpression of C-terminal truncations in mice models. However, these models cannot fully recapitulate the pathogenesis of myeloid malignancies. Patient-derived induced pluripotent stem cells (iPSCs) provide valuable disease models that allow us to understand disease-related molecular pathways and develop novel targeted therapies. Here, we generated iPSCs from a patient with myeloproliferative neoplasm carrying a heterozygous ASXL1 mutation. The iPSCs we generated exhibited the morphology of pluripotent cells, highly expressed pluripotent markers, excellent differentiation potency in vivo, and normal karyotype. Subsequently, iPSCs with or without ASXL1 mutation were induced to differentiate into hematopoietic stem/progenitor cells, and we found that ASXL1 mutation led to myeloid-biased output and impaired erythroid differentiation. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that terms related to embryonic development, myeloid differentiation, and immune- and neural-related processes were most enriched in the differentially expressed genes. Western blot demonstrated that the global level of H2AK119ub was significantly decreased when mutant ASXL1 was present. Chromatin Immunoprecipitation Sequencing showed that most genes associated with stem cell maintenance were upregulated, whereas occupancies of H2AK119ub around these genes were significantly decreased. Thus, the iPSC model carrying ASXL1 mutation could serve as a potential tool to study the pathogenesis of myeloid malignancies and to screen targeted therapy for patients. [ABSTRACT FROM AUTHOR]

The BAF chromatin remodeler regulates lineage commitment including cranial neural crest cell (CNCC) specification. Variants in BAF subunits cause Coffin-Siris syndrome (CSS), a congenital disorder characterized by coarse craniofacial features and intellectual disability. Approximately 50% of individuals with CSS harbor variants in one of the mutually exclusive BAF subunits, ARID1A/ARID1B. While Arid1a deletion in mouse neural crest causes severe craniofacial phenotypes, little is known about the role of ARID1A in CNCC specification. Using CSS-patient-derived ARID1A +/− induced pluripotent stem cells to model CNCC specification, we discovered that ARID1A -haploinsufficiency impairs epithelial-to-mesenchymal transition (EMT), a process necessary for CNCC delamination and migration from the neural tube. Furthermore, wild-type ARID1A-BAF regulates enhancers associated with EMT genes. ARID1A-BAF binding at these enhancers is impaired in heterozygotes while binding at promoters is unaffected. At the sequence level, these EMT enhancers contain binding motifs for ZIC2, and ZIC2 binding at these sites is ARID1A-dependent. When excluded from EMT enhancers, ZIC2 relocates to neuronal enhancers, triggering aberrant neuronal gene activation. In mice, deletion of Zic2 impairs NCC delamination, while ZIC2 overexpression in chick embryos at post-migratory neural crest stages elicits ectopic delamination from the neural tube. These findings reveal an essential ARID1A-ZIC2 axis essential for EMT and CNCC delamination. [Display omitted] ARID1A modulates chromatin accessibility at enhancers of genes required for epithelial-to-mesenchymal transition, a process essential for cranial neural crest cell (CNCC) specification. Haploinsufficiency of ARID1A attenuates ZIC2 binding at these enhancers, resulting in impaired CNCC formation with an aberrant neuronal trajectory. This study reveals an ARID1A-ZIC2 axis essential for CNCC specification. [ABSTRACT FROM AUTHOR]

The generation of stem cell–derived β-like cells (sBCs) holds promise as not only an abundant insulin-producing cell source for replacement therapy of type 1 diabetes (T1D) but also as an invaluable model system for investigating human β-cell development, immunogenicity, and function. Several groups have developed methodology to direct differentiate human pluripotent stem cells into pancreatic cell populations that include glucose-responsive sBCs. Nevertheless, the process of generating sBCs poses substantial experimental challenges. It involves lengthy differentiation periods, there is substantial variability in efficiency, and there are inconsistencies in obtaining functional sBCs. Here, we describe a simple and effective cryopreservation approach for sBC cultures that yields homogeneous sBC clusters that are enriched for insulin-expressing cells while simultaneously depleting proliferative progenitors. Thawed sBCs have enhanced glucose-stimulated insulin release compared with controls in vitro and can effectively engraft and function in vivo. Collectively, this approach alleviates current challenges with inefficient and variable sBC generation while improving their functional state. We anticipate that these findings can inform ongoing clinical application of sBCs for the treatment of patients with T1D and serve as an important resource for the wider diabetes field that will allow for accelerated research discoveries. Article Highlights: Human stem cell–derived β-like cells (sBCs) are a vital tool for the field of diabetes research but are technically difficult to produce and challenging to maintain in culture. We describe a new cost-effective and simple approach to cryopreserving differentiated sBCs. Frozen and thawed (F/T) sBCs are more uniform, display an enriched β-cell fraction, and have improved function while depleting SOX9+ progenitors. F/T sBCs can successfully engraft and display stimulated insulin release in the absence of any cystic structures. Cryopreservation will enable more researchers to use sBCs for complex experiments to investigate human diabetes pathogenesis. [ABSTRACT FROM AUTHOR]

Human induced pluripotent stem cell-derived models are a well-established preclinical tool, with the ability to retain the genetics of the individual from which they are derived. Here we comment on the global representation and accessibility of such cellular tools from African population groups. [ABSTRACT FROM AUTHOR]

Inner ear organoids play a crucial role in hearing research. In comparison to other animal models and 2D cell culture systems, inner ear organoids offer significant advantages for studying the mechanisms of inner ear development and exploring novel approaches to disease treatment. Inner ear organoids derived from human cells are more closely resemble normal human organs in development and function. The 3D culture system of the inner ear organoid enhances cell–cell interactions and mimics the internal environment. In this review, the progress and limitations of organoid culture methods derived from tissue‐specific progenitors and pluripotent stem cells (PSCs) are summarized, which may offer new insights into generating organoids that closely resemble the inner ear in terms of morphology and function. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells (PSCs) offer many potential research and clinical benefits due to their ability to differentiate into nearly every cell type in the body. They are often used as model systems to study early stages of ontogenesis to better understand key developmental pathways, as well as for drug screening. However, in order to fully realise the potential of PSCs and their translational applications, a deeper understanding of developmental pathways, especially in humans, is required. Several signalling molecules play important roles during development and are required for proper differentiation of PSCs. The concentration and timing of signal activation are important, with perturbations resulting in improper development and/or pathology. Bone morphogenetic proteins (BMPs) are one such key group of signalling molecules involved in the specification and differentiation of various cell types and tissues in the human body, including those related to tooth and otic development. In this review, we describe the role of BMP signalling and its regulation, the consequences of BMP dysregulation in disease and differentiation, and how PSCs can be used to investigate the effects of BMP modulation during development, mainly focusing on otic development. Finally, we emphasise the unique role of BMP4 in otic specification and how refined understanding of controlling its regulation could lead to the generation of more robust and reproducible human PSC-derived otic organoids for research and translational applications. [ABSTRACT FROM AUTHOR]

We describe the development of a bioink to bioprint human induced pluripotent stem cells (hiPSCs) for possible cardiac tissue engineering using a gelatin methacrylate (GelMA)-based hydrogel. While previous studies have shown that GelMA at a low concentration (5% w/v) allows for the growth of diverse cells, its 3D printability has been found to be limited by its low viscosity. To overcome that drawback, making the hydrogel both compatible with hiPSCs and 3D-printable, we developed an extrudable GelMA-based bioink by adding xanthan gum (XG). The GelMA-XG composite hydrogel had an elastic modulus (~9 kPa) comparable to that of cardiac tissue, and enabled 3D printing with high values of printing accuracy (83%) and printability (0.98). Tests with hiPSCs showed the hydrogel's ability to promote their proliferation within both 2D and 3D cell cultures. The tests also showed that hiPSCs inside hemispheres of the hydrogel were able to differentiate into cardiomyocytes, capable of spontaneous contractions (average frequency of ~0.5 Hz and amplitude of ~2%). Furthermore, bioprinting tests proved the possibility of fabricating 3D constructs of the hiPSC-laden hydrogel, with well-defined line widths (~800 μm). [ABSTRACT FROM AUTHOR]

Implant stability can be compromised by factors such as inadequate bone quality and infection, leading to potential implant failure. Ensuring implant stability and longevity is crucial for patient satisfaction and quality of life. In this multicenter, randomized, double-blind clinical trial, we assessed the impact of a bone bioactive liquid (BBL) on the Galaxy TS implant's performance, stability, and osseointegration. We evaluated the impact stability, osseointegration, and pain levels using initial stability quotient (ISQ) measurements, CBCT scans, and pain assessment post-surgery. Surface analysis was performed using scanning electron microscopy (SEM) and atomic force microscopy (AFM). In vitro studies examined the BBL's effects on dental pulp pluripotent stem cells' (DPPSCs') osteogenesis and inflammation modulation in human macrophages. All implants successfully osseointegrated, as demonstrated by the results of our clinical and histological studies. The BBL-treated implants showed significantly lower pain scores by day 7 (p < 0.00001) and improved stability by day 30 (ISQ > 62.00 ± 0.59, p < 8 × 10−7). By day 60, CBCT scans revealed an increased bone area ratio in BBL-treated implants. AFM images demonstrated the BBL's softening and wettability effect on implant surfaces. Furthermore, the BBL promoted DPPSCs' osteogenesis and modulated inflammatory markers in human primary macrophages. This study presents compelling clinical and biological evidence that BBL treatment improves Galaxy TS implant stability, reduces pain, and enhances bone formation, possibly through surface tension modulation and immunomodulatory effects. This advancement holds promise for enhancing patient outcomes and implant longevity. [ABSTRACT FROM AUTHOR]

The effectiveness and safety of mesenchymal stem cell (MSC) therapy have been substantiated across various diseases. Nevertheless, challenges such as the restricted in vitro expansion capacity of tissue‐derived MSCs and the clinical instability due to the high heterogeneity of isolated cells require urgent resolution. The induced pluripotent stem cell‐derived MSCs (iPSC‐MSCs), which is differentiated from iPSCs via specific experimental pathways, holds considerable potential as a substitute for tissue derived MSCs. Multiple studies have demonstrated that iPSCs can be differentiated into iPSC‐MSCs through diverse differentiation strategies. Research suggests that iPSC‐MSCs, when compared to tissue derived MSCs, exhibit superior characteristics in terms of proliferation ability, immune modulation capacity, and biological efficiency. In this review, we meticulously described and summarized the experimental methods of iPSC differentiation into iPSC‐MSCs, the application of iPSC‐MSCs in various disease models, the latest advancements in clinically relevant iPSC‐derived cell products, and the development strategies for the next generation of iPSC‐derived therapy products (not only cell products but also their derivatives). [ABSTRACT FROM AUTHOR]

Background: SETBP1 Haploinsufficiency Disorder (SETBP1-HD) is characterised by mild to moderate intellectual disability, speech and language impairment, mild motor developmental delay, behavioural issues, hypotonia, mild facial dysmorphisms, and vision impairment. Despite a clear link between SETBP1 mutations and neurodevelopmental disorders the precise role of SETBP1 in neural development remains elusive. We investigate the functional effects of three SETBP1 genetic variants including two pathogenic mutations p.Glu545Ter and SETBP1 p.Tyr1066Ter, resulting in removal of SKI and/or SET domains, and a point mutation p.Thr1387Met in the SET domain. Methods: Genetic variants were introduced into induced pluripotent stem cells (iPSCs) and subsequently differentiated into neurons to model the disease. We measured changes in cellular differentiation, SETBP1 protein localisation, and gene expression changes. Results: The data indicated a change in the WNT pathway, RNA polymerase II pathway and identified GATA2 as a central transcription factor in disease perturbation. In addition, the genetic variants altered the expression of gene sets related to neural forebrain development matching characteristics typical of the SETBP1-HD phenotype. Limitations: The study investigates changes in cellular function in differentiation of iPSC to neural progenitor cells as a human model of SETBP1 HD disorder. Future studies may provide additional information relevant to disease on further neural cell specification, to derive mature neurons, neural forebrain cells, or brain organoids. Conclusions: We developed a human SETBP1-HD model and identified perturbations to the WNT and POL2RA pathway, genes regulated by GATA2. Strikingly neural cells for both the SETBP1 truncation mutations and the single nucleotide variant displayed a SETBP1-HD-like phenotype. [ABSTRACT FROM AUTHOR]

In the present study, we aimed to establish and characterize a mature cortical spheroid model system for Kleefstra syndrome (KS) using patient-derived iPSC. We identified key differences in the growth behavior of KS spheroids determined by reduced proliferation marked by low Ki67 and high E-cadherin expression. Conversely, in the spheroid-based neurite outgrowth assay KS outperformed the control neurite outgrowth due to higher BDNF expression. KS spheroids were highly enriched in VGLUT1/2-expressing glutamatergic and ChAT-expressing cholinergic neurons, while TH-positive catecholamine neurons were significantly underrepresented. Furthermore, high NMDAR1 expression was also detected in the KS spheroid, similarly to other patients-derived neuronal cultures, denoting high NMDAR1 expression as a general, KS-specific marker. Control and KS neuronal progenitors and neurospheres were exposed to different toxicants (paraquat, rotenone, bardoxolone, and doxorubicin), and dose-response curves were assessed after acute exposure. Differentiation stage and compound-specific differences were detected with KS neurospheres being the most sensitive to paraquat. Altogether this study describes a robust 3D model system expressing the disease-specific markers and recapitulating the characteristic pathophysiological traits. This platform is suitable for testing developing brain-adverse environmental effects interactions, drug development, and screening towards individual therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: Human epithelium-derived stem cells and induced pluripotent stem cells (hiPSCs) possess the capability to support tooth formation and differentiate into functional enamel-secreting ameloblasts, making them promising epithelial-component substitutes for future human tooth regeneration. However, current tissue recombination approaches are not only technically challenging, requiring precise induction procedures and sophisticated microsurgery, but also exhibit low success rates in achieving tooth formation and ameloblastic differentiation. Methods: Suspended human keratinocyte stem cells (hKSCs) or cells from three hiPSC lines were directly mixed with dissociated embryonic mouse dental mesenchymal cells (mDMCs) that possess odontogenic potential in different proportions and reaggregated them to construct bioengineered tooth germs. The success rates of tooth formation and ameloblastic differentiation were confirmed after subrenal culture. The sorting capability, sequential development, and ameloblastic differentiation of stem cells were examined via GFP tracing, RT-PCR, and histological analysis, respectively. Results: Our reaggregation approach achieved an impressive success rate of more than 90% in tooth formation and 100% in ameloblastic differentiation when the chimeric tooth germs contained 1%~10% hKSCs or 5% hiPSCs. In addition, we observed that hiPSCs, upon exposure to mDMCs, initially transformed into epidermal cells, as indicated by KRT14 and CD29 expression, before progressing into dental epithelial cells, as indicated by SP6 and SHH expression. We also found that epithelial-derived hiPSCs, when reaggregated with mDMCs, were more favorable for tooth formation than their mesenchymal-derived counterparts. Conclusions: This study establishes a simplified yet highly effective cell-cell reaggregation strategy for inducing stem cells to support tooth formation and differentiate into functional ameloblasts, paving the way for novel approaches for the development of stem cell-based tooth organoids and bioengineered tooth germs in vitro. [ABSTRACT FROM AUTHOR]

Mural cells are central to vascular integrity and function. In this study, we demonstrate the innovative use of the transcription factor NKX3.1 to guide the differentiation of human induced pluripotent stem cells into mural progenitor cells (iMPCs). By transiently activating NKX3.1 in mesodermal intermediates, we developed a method that diverges from traditional growth factor-based differentiation techniques. This approach efficiently generates a robust iMPC population capable of maturing into diverse functional mural cell subtypes, including smooth muscle cells and pericytes. These iMPCs exhibit key mural cell functionalities such as contractility, deposition of extracellular matrix, and the ability to support endothelial cell-mediated vascular network formation in vivo. Our study not only underscores the fate-determining significance of NKX3.1 in mural cell differentiation but also highlights the therapeutic potential of these iMPCs. We envision these insights could pave the way for a broader use of iMPCs in vascular biology and regenerative medicine. Mural progenitor cells are crucial for vascular stability. Here, the authors generate these cells from human pluripotent stem cells using NKX3.1 and show that they can mature into various mural cell types and contribute to blood vessel formation. [ABSTRACT FROM AUTHOR]

To enhance the practical application of intestinal organoids, it is imperative to establish standardized guidelines. This proposed standardization outlines a comprehensive framework to ensure consistency and reliability in the development, characterization, and application of intestinal organoids. The recommended guidelines encompass crucial parameters, including culture conditions, critical quality attributes, quality control measures, and functional assessments, aimed at fostering a standardized approach across diverse research initiatives. The implementation of these guidelines is anticipated to significantly contribute to the reproducibility and comparability of results in the burgeoning field of intestinal organoid research. [ABSTRACT FROM AUTHOR]