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BACKGROUND: Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation. METHODS: Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC). RESULTS: We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1+ lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1+ lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation. CONCLUSION: Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro.

Our understanding of the transitions of human embryonic stem cells (hESCs) between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of hESCs as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for formation of the actin ring, to establish uniform cell mechanics within naïve colonies, to promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and for effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

Tissue engineering can provide in vitro models for drug testing, disease modeling, and perhaps someday, tissue/organ replacements. For building 3D heart tissue, the alignment of cardiac cells or cardiomyocytes (CMs) is important in generating a synchronously contracting tissue. To that end, researchers have generated several fabrication methods for building heart tissue, but direct comparisons of pros and cons using the same cell source is lacking. Here, we derived cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) and compare the assembly of these cells using three fabrication methods: cardiospheres, muscle rings, and muscle strips. All three protocols successfully generated compacted tissue comprised of hiPSC-derived CMs stable for at least 2 weeks. The percentage of aligned cells was greatest in the muscle strip (55%) and the muscle ring (50%) compared with the relatively unaligned cardiospheres (35%). The iPSC-derived CMs within the muscle strip also exhibited the greatest elongation, with elongation factor at 2.0 compared with 1.5 for the muscle ring and 1.2 for the cardiospheres. This is the first direct comparison of various fabrication techniques using the same cell source.

The developmental origin of blood-forming hematopoietic stem cells (HSCs) is a longstanding question. Here, our non-invasive genetic lineage tracing in mouse embryos pinpoints that artery endothelial cells generate HSCs. Arteries are transiently competent to generate HSCs for 2.5 days (∼E8.5-E11) but subsequently cease, delimiting a narrow time frame for HSC formation in vivo. Guided by the arterial origins of blood, we efficiently and rapidly differentiate human pluripotent stem cells (hPSCs) into posterior primitive streak, lateral mesoderm, artery endothelium, hemogenic endothelium, and >90% pure hematopoietic progenitors within 10 days. hPSC-derived hematopoietic progenitors generate T, B, NK, erythroid, and myeloid cells in vitro and, critically, express hallmark HSC transcription factors HLF and HOXA5-HOXA10, which were previously challenging to upregulate. We differentiated hPSCs into highly enriched HLF+ HOXA+ hematopoietic progenitors with near-stoichiometric efficiency by blocking formation of unwanted lineages at each differentiation step. hPSC-derived HLF+ HOXA+ hematopoietic progenitors could avail both basic research and cellular therapies.

BACKGROUND: A traditional view is that stem cells (SCs) divide slowly. Meanwhile, both embryonic and pluripotent SCs display a shorter cell cycle duration (CCD) in comparison to more committed progenitors (CPs). METHODS: We examined the in vitro proliferation and cycling behavior of somatic adult human cells using live cell imaging of passage zero keratinocytes and single-cell RNA sequencing. RESULTS: We found two populations of keratinocytes: those with short CCD and protracted near exponential growth, and those with long CCD and terminal differentiation. Applying the ergodic principle, the comparative numbers of cycling cells in S phase in an enriched population of SCs confirmed a shorter CCD than CPs. Further, analysis of single-cell RNA sequencing of cycling adult human keratinocyte SCs and CPs indicated a shortening of both G1 and G2M phases in the SC. CONCLUSIONS: Contrary to the pervasive paradigm, SCs progress through cell cycle more quickly than more differentiated dividing CPs. Thus, somatic human adult keratinocyte SCs may divide infrequently, but divide rapidly when they divide. Additionally, it was found that SC-like proliferation persisted in vitro.

Age remains the central risk factor for many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Although the mechanisms of aging are complex, the age-related accumulation of senescent cells in neurodegeneration is well documented and their clearance can alleviate disease-related features in preclinical models. Senescence-like characteristics are observed in both neuronal and glial lineages, but their relative contribution to aging and neurodegeneration remains unclear. Human pluripotent stem cell-derived neurons provide an experimental model system to induce neuronal senescence. However, the extensive heterogeneity in the profile of senescent neurons and the methods to assess senescence remain major challenges. Here, we review the evidence of cellular senescence in neuronal aging and disease, discuss human pluripotent stem cell-based model systems used to investigate neuronal senescence and propose a panel of cellular and molecular hallmarks to characterize senescent neurons. Understanding the role of neuronal senescence may yield novel therapeutic opportunities in neurodegenerative disease.

Human pluripotent stem cell-derived neural progenitor cells (NPCs) are an essential tool for the study of brain development and developmental disorders such as autism. Here, we present a protocol to generate NPCs rapidly and reproducibly from human stem cells using dual-SMAD inhibition coupled with a brief pulse of mouse neurogenin-2 (Ngn2) overexpression. We detail the 48-h induction scheme deployed to produce these cells-termed stem cell-derived Ngn2-accelerated progenitor cells-followed by steps for expansion, purification, banking, and quality assessment. For complete details on the use and execution of this protocol, please refer to Wells et al.1.

Brain mural cells regulate development and function of the blood-brain barrier and control blood flow. Existing in vitro models of human brain mural cells have low expression of key mural cell genes, including NOTCH3. Thus, we asked whether activation of Notch3 signaling in hPSC-derived neural crest could direct the differentiation of brain mural cells with an improved transcriptional profile. Overexpression of the Notch3 intracellular domain (N3ICD) induced expression of mural cell markers PDGFRβ, TBX2, FOXS1, KCNJ8, SLC6A12, and endogenous Notch3. The resulting N3ICD-derived brain mural cells produced extracellular matrix, self-assembled with endothelial cells, and had functional KATP channels. ChIP-seq revealed that Notch3 serves as a direct input to relatively few genes in the context of this differentiation process. Our work demonstrates that activation of Notch3 signaling is sufficient to direct the differentiation of neural crest to mural cells and establishes a developmentally relevant differentiation protocol.

Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations.

Primordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. They transmit genetic and epigenetic information across generations. Given the prominent role of germline defects in diseases such as infertility, detailed understanding of human PGC (hPGC) development has important implications in reproductive medicine and studying human evolution. Yet, hPGC specification remains an elusive process. Here, we report the induction of hPGC-like cells (hPGCLCs) in a bioengineered human pluripotent stem cell (hPSC) culture that mimics peri-implantation human development. In this culture, amniotic ectoderm-like cells (AMLCs), derived from hPSCs, induce hPGCLC specification from hPSCs through paracrine signaling downstream of ISL1. Our data further show functional roles of NODAL, WNT, and BMP signaling in hPGCLC induction. hPGCLCs are successfully derived from eight non-obstructive azoospermia (NOA) participant-derived hPSC lines using this biomimetic platform, demonstrating its promise for screening applications.

Cardiac cellular fate transition holds remarkable promise for the treatment of ischemic heart disease. We report that overexpressing two transcription factors, Sall4 and Gata4, which play distinct and overlapping roles in both pluripotent stem cell reprogramming and embryonic heart development, induces a fraction of stem-like cells in rodent cardiac fibroblasts that exhibit unlimited ex vivo expandability with clonogenicity. Transcriptomic and phenotypic analyses reveal that around 32 ± 6.4% of the expanding cells express Nkx2.5, while 13 ± 3.6% express Oct4. Activated signaling pathways like PI3K/Akt, Hippo, Wnt, and multiple epigenetic modification enzymes are also detected. Under suitable conditions, these cells demonstrate a high susceptibility to differentiating into cardiomyocyte, endothelial cell, and extracardiac neuron-like cells. The presence of partially pluripotent-like cells is characterized by alkaline phosphatase staining, germ layer marker expression, and tumor formation in injected mice (n = 5). Additionally, significant stem-like fate transitions and cardiogenic abilities are induced in human cardiac fibroblasts, but not in rat or human skin fibroblasts. Molecularly, we identify that SALL4 and GATA4 physically interact and synergistically stimulate the promoters of pluripotency genes but repress fibrogenic gene, which correlates with a primitive transition process. Together, this study uncovers a new cardiac regenerative mechanism that could potentially advance therapeutic endeavors and tissue engineering. [ABSTRACT FROM AUTHOR]

Background: Adoptive immunotherapy using natural killer (NK) cells has attracted considerable interest in numerous clinical trials targeting both hematological and solid tumors. Traditionally, NK cells are primarily derived from either peripheral blood (PB) or umbilical cord blood (UCB). However, these methods can lead to variability and heterogeneity within the NK cell population. In contrast, induced pluripotent stem cell (iPSC)-derived NK (iNK) cells provide a more controlled and uniform cellular population, suitable for large-scale clinical applications. This makes iNK cells a promising option for developing "off-the-shelf" immunotherapeutic products. Nevertheless, current NK cell differentiation protocols, which rely on embryoid body (EB) cultures, are labor-intensive and susceptible to unwanted heterogeneity during differentiation. Here, we developed a more efficient approach for generating iNK cells by employing a monolayer and feeder-free differentiation protocol, alongside optimized culture media. Methods: The iNK cells were generated using a two-step in vitro monolayer feeder-free system following NK cell development. To evaluate their maturity, phenotypic analysis was performed using flow cytometry, comparing with PB-NK cells and the NK-92 cell line. Additionally, single-cell RNA sequencing was performed to examine their transcriptomic profiles. The cytotoxic activity of the iNK cells was evaluated by co-culturing with cholangiocarcinoma (CCA) and breast cancer (BCA) cell lines in both monolayer (2D) and tumor spheroid (3D) co-culture systems. Results: We successfully differentiated iPSCs into mesoderm (ME), hematopoietic stem/progenitor cells (HSPCs), and NK cells. The resulting iNK cells exhibited typical NK cell markers such as CD45, CD56, and CD16, and expressed key functional proteins, including both activating and inhibitory receptors. Single-cell RNA sequencing confirmed that the transcriptomic profile of our iNK cells closely resembles that of PB-NK cells. Importantly, our iNK cells demonstrated strong cytotoxic abilities against various CCA and BCA cell lines, surpassing the NK-92 cell line in both monolayer cultures and tumor spheroid cultures. Conclusion: This study highlights the potential of iPSCs as an effective alternative cell source for generating NK cells. Using a two-step in vitro monolayer feeder-free system, we successfully generated iNK cells that not only expressed key NK cell markers and their receptors but also displayed a transcriptomic profile closely resembling PB-NK cells. Furthermore, iNK cells exhibited cytotoxicity against CCA and BCA cell lines comparable to that of PB-NK cells. This approach could pave the way for off-the-shelf NK cell products, potentially enhancing the effectiveness of adoptive NK cell therapy. [ABSTRACT FROM AUTHOR]

As crucial phagocytes of the innate immune system, macrophages (Mϕs) protect mammalian hosts, maintain tissue homeostasis and influence disease pathogenesis. Nonetheless, Mϕs are susceptible to various pathogens, including bacteria, viruses and parasites, which cause various infectious diseases, necessitating a deeper understanding of pathogen–Mϕ interactions and therapeutic insights. Pluripotent stem cells (PSCs) have been efficiently differentiated into PSC‐derived Mϕs (PSCdMϕs) resembling primary Mϕs, advancing the modelling and cell therapy of infectious diseases. However, the mass production of PSCdMϕs, which lack proliferative capacity, relies on large‐scale expansions of PSCs, thereby increasing both costs and culture cycles. Notably, Mϕs deficient in the MafB/c‐Maf genes have been reported to re‐enter the cell cycle with the stimulation of specific growth factor cocktails, turning into self‐renewing Mϕs (SRMϕs). This review summarizes the applications of PSCdMϕs in the modelling and cell therapy of infectious diseases and strategies for establishing SRMϕs. Most importantly, we innovatively propose that PSCs can serve as a gene editing platform to creating PSC‐derived SRMϕs (termed PSRMϕs), addressing the resistance of Mϕs against genetic manipulation. We discuss the challenges and possible solutions in creating PSRMϕs. In conclusion, this review provides novel insights into the development of physiologically relevant and expandable Mϕ models, highlighting the enormous potential of PSRMϕs as a promising avenue for the modelling and cell therapy of infectious diseases. [ABSTRACT FROM AUTHOR]

Introduction: In recent years, high-density microelectrode arrays (HD-MEAs) have emerged as a valuable tool in preclinical research for characterizing the electrophysiology of human induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs). HD-MEAs enable the capturing of both extracellular and intracellular signals on a large scale, while minimizing potential damage to the cell. However, despite technological advancements of HD-MEAs, there is a lack of effective data-analysis platforms that are capable of processing and analyzing the data, particularly in the context of cardiac arrhythmias and drug testing. Methods: To address this need, we introduce CardioMEA, a comprehensive data-analysis platform designed specifically for HD-MEA data that have been obtained from iPSCCMs. CardioMEA features scalable data processing pipelines and an interactive web-based dashboard for advanced visualization and analysis. In addition to its core functionalities, CardioMEA incorporates modules designed to discern crucial electrophysiological features between diseased and healthy iPSC-CMs. Notably, CardioMEA has the unique capability to analyze both extracellular and intracellular signals, thereby facilitating customized analyses for specific research tasks. Results and discussion: We demonstrate the practical application of CardioMEA by analyzing electrophysiological signals from iPSC-CM cultures exposed to seven antiarrhythmic drugs. CardioMEA holds great potential as an intuitive, userfriendly platform for studying cardiac diseases and assessing drug effects. [ABSTRACT FROM AUTHOR]

The combination of aminophylline and salbutamol is frequently used in clinical practice in the treatment of obstructive lung diseases. While the side effects (including arrhythmias) of the individual bronchodilator drugs were well described previously, the side effects of combined treatment are almost unknown. We aimed to study the arrhythmogenic potential of combined aminophylline and salbutamol treatment in vitro. For this purpose, we used the established atomic force microscopy (AFM) model coupled with cardiac organoids derived from human pluripotent stem cells (hPSC-CMs). We focused on the chronotropic, inotropic, and arrhythmogenic effects of salbutamol alone and aminophylline and salbutamol combined treatment. We used a method based on heart rate/beat rate variability (HRV/BRV) analysis to detect arrhythmic events in the hPSC-CM based AFM recordings. Salbutamol and aminophylline had a synergistic chronotropic and inotropic effect compared to the effects of monotherapy. Our main finding was that salbutamol reduced the arrhythmogenic effect of aminophylline, most likely mediated by endothelial nitric oxide synthase activated by beta-2 adrenergic receptors. These findings were replicated and confirmed using hPSC-CM derived from two cell lines (CCTL4 and CCTL12). Data suggest that salbutamol as an add-on therapy may not only deliver a bronchodilator effect but also increase the cardiovascular safety of aminophylline, as salbutamol reduces its arrhythmogenic potential. [ABSTRACT FROM AUTHOR]

Obesity, a growing global health concern, is closely linked to depression. However, the neural mechanism of association between obesity and depression remains poorly understood. In this study, neural‐specific WFS1 deficiency exacerbates the vicious cycle of obesity and depression in mice fed a high‐fat diet (HFD), positioning WFS1 as a crucial factor in this cycle. Through human pluripotent stem cells (hESCs) neural differentiation, it is demonstrated that WFS1 regulates Zn2+ homeostasis and the apoptosis of neural progenitor cells (NPCs) and cerebral organoids by inhibiting the zinc transporter ZnT3 under the situation of dysregulated lipid metabolism. Notably, riluzole regulates ZnT3 expression to maintain zinc homeostasis and protect NPCs from lipotoxicity‐induced cell death. Importantly, riluzole, a therapeutic molecule targeting the nervous system, in vivo administration prevents HFD‐induced obesity and associated depression. Thus, a WFS1‐ZnT3‐Zn2+ axis critical is demonstrated for the vicious cycle of obesity and depression and that riluzole may have the potential to reverse this process against obesity and depression. [ABSTRACT FROM AUTHOR]

Microtubule affinity-regulating kinase 2 (MARK2) contributes to establishing neuronal polarity and developing dendritic spines. Although large-scale sequencing studies have associated MARK2 variants with autism spectrum disorder (ASD), the clinical features and variant spectrum in affected individuals with MARK2 variants, early developmental phenotypes in mutant human neurons, and the pathogenic mechanism underlying effects on neuronal development have remained unclear. Here, we report 31 individuals with MARK2 variants and presenting with ASD, other neurodevelopmental disorders, and distinctive facial features. Loss-of-function (LoF) variants predominate (81%) in affected individuals, while computational analysis and in vitro expression assay of missense variants supported the effect of MARK2 loss. Using proband-derived and CRISPR-engineered isogenic induced pluripotent stem cells (iPSCs), we show that MARK2 loss leads to early neuronal developmental and functional deficits, including anomalous polarity and dis-organization in neural rosettes, as well as imbalanced proliferation and differentiation in neural progenitor cells (NPCs). Mark2 +/− mice showed abnormal cortical formation and partition and ASD-like behavior. Through the use of RNA sequencing (RNA-seq) and lithium treatment, we link MARK2 loss to downregulation of the WNT/β-catenin signaling pathway and identify lithium as a potential drug for treating MARK2 -associated ASD. [Display omitted] MARK2 plays essential roles in the central nervous system. Here, we demonstrate the association between MARK2 variants, downregulated WNT/β-catenin signaling, and autism spectrum disorder (ASD) through the study of individuals with ASD and Mark2 +/− mice. [ABSTRACT FROM AUTHOR]

The demand for skin models as alternatives to animal testing has grown due to ethical concerns and the need for accurate substance evaluation. These alternatives, known as New Approach Methodologies (NAMs), are increasingly used for regulatory decisions. Current skin models from primary human cells often rely on bovine collagen, raising ethical issues. This study explores self-assembled skin models (SASM) as a new method, utilizing hair follicle-derived keratinocytes reprogrammed into induced pluripotent stem cells (iPSC) and differentiated into fibroblasts and keratinocytes. The model relies on the ability of fibroblasts to secrete collagen to produce a xeno-free dermal layer and on the differentiation of keratinocytes to create a functional epidermal layer. These layers exhibited confirmed metabolic activity and the capability to withstand test substances. The successful development of SASM underscores the significance of accurate alternatives in dermatological research, providing an ethical and reliable option for substance evaluation and regulatory testing. [ABSTRACT FROM AUTHOR]

Objective: We aimed to evaluate the efficacy of stem cell-derived exosomes for treating ischemic stroke and to screen for the optimal administration strategy. Methods: We searched PubMed, Web of Science, Embase, Cochrane Library, and Scopus databases for relevant studies published from their inception to 31 December 2023. Conventional and network meta-analyses of the routes of administration, types, and immune compatibility of stem cell-derived exosomes were performed using the cerebral infarct volume (%) and modified neurological severity score (mNSS) as outcome indicators. Results: A total of 38 randomized controlled animal experiments were included. Conventional meta-analysis showed that compared with the negative control group: intravenous administration significantly reduced the cerebral infarct volume (%) and mNSS; intranasal administration significantly reduced the cerebral infarct volume (%); and intracerebral administration significantly reduced the mNSS. Adipose-derived mesenchymal stem cell-derived exosomes (ADSC-Exos), bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos), dental pulp stem cell-derived exosomes (DPSC-Exos) and neural stem cell-derived exosomes (NSC-Exos) significantly reduced the cerebral infarct volume (%) and mNSS; Endothelial progenitor cell-derived exosomes (EPC-Exos), embryonic stem cell-derived exosomes (ESC-Exos), induced pluripotent stem cell-derived exosomes (iPSC-Exos) and neural progenitor cell-derived exosomes (NPC-Exos) significantly reduced the cerebral infarct volume (%); Umbilical cord mesenchymal stem cell-derived exosomes (UCMSC-Exos) significantly reduced the mNSS; and there was no significant difference between urogenital stem cell-derived exosomes (USC-Exos) and negative controls. Engineered modified exosomes had better efficacy than unmodified exosomes. Both allogeneic and xenogeneic stem cell-derived exosomes significantly reduced the cerebral infarct volume (%) and the mNSS. The network meta-analysis showed that intravenous administration was the best route of administration for reducing the cerebral infarct volume (%) and mNSS. Among the 10 types of stem cell-derived exosomes that were administered intravenously, BMSC-Exos were the best type for reducing the cerebral infarct volume (%) and the mNSS. Allogeneic exosomes had the best efficacy in reducing the cerebral infarct volume (%), whereas xenogeneic stem cell-derived exosomes had the best efficacy in reducing the mNSS. Conclusion: This meta-analysis, by integrating the available evidence, revealed that intravenous administration is the best route of administration, that BMSC-Exos are the best exosome type, that allogeneic exosomes have the best efficacy in reducing the cerebral infarct volume (%), and that xenogeneic exosomes have the best efficacy in reducing mNSS, which can provide options for preclinical studies. In the future, more high-quality randomized controlled animal experiments, especially direct comparative evidence, are needed to determine the optimal administration strategy for stem cell-derived exosomes for ischemic stroke. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/display%5frecord.php?ID=CRD42024497333, PROSPERO, CRD42024497333 [ABSTRACT FROM AUTHOR]

Besides being an essential structural component of plasma membranes and the precursor of many functional compounds and signaling molecules, cholesterol was also proposed to play a role in the etiology and/or manifestation of Parkinson's disease (PD). However, so far systematic investigations on the role of cholesterol and its metabolites present in the brain for the etiology of PD are missing. Here, we investigate for the first time the association of PD with SNPs in the genes of four cytochromes P450 (P450), CYP46A1, CYP39A1, CYP27A1 and CYP7B1, which are critical for the degradation of cholesterol in the brain. Analyzing 1,349 individuals from the PPMI data base, we found 24 SNPs in these four genes, which are significantly over- or under-represented in patients suffering from idiopathic PD (IPD). Studying each of the 362 IPD patients individually, we found that most patients (45%) showed only one associated SNP in one of the four P450 genes, while 31% displayed two associated SNPs and 18% three associated SNPs. The occurrence of some associated SNPs is in the same order of magnitude as SNPs in the GBA (beta-glucocerebrosidase) and thus might reflect a genetic predisposition for PD. As all 24 SNPs were located in introns and 3′ untranslated regions, we evaluated the prospective regulatory impact of the surrounding genomic regions by using transcriptome and epigenome data from the Foundational Data Initiative for Parkinson Disease (FOUNDIN-PD). FOUNDIN-PD provides gene expression, open chromatin and DNA methylation data in a cohort of 89 induced pluripotent stem cell (iPSC) lines differentiated to dopaminergic (DA) neurons derived from people in the PPMI study. Indeed, two of the 24 SNPs, one in CYP7B1 (rs118111353) and the other one in CYP27A1 (rs74446825), were localized within a region of open chromatin in differentiated neurons. Interestingly, all iPSC lines with open chromatin in rs118111353 showed the reference allele. As all four P450, CYP46A1, CYP39A1, CYP27A1 and CYP7B1, are expressed in dopaminergic neurons, we discuss further functional studies to connect SNPs in regulatory regions with gene expression levels. Finally, potential possibilities for personalized therapeutic treatment of patients with SNPs in the four investigated P450 are discussed. [ABSTRACT FROM AUTHOR]

Self‐organizing brain organoids provide a promising tool for studying human development and disease. Here we created human forebrain organoids with stable and homogeneous expression of channelrhodopsin‐2 (ChR2) by generating AAVS1 safe harbor locus‐targeted, ChR2 knocked‐in human pluripotent stem cells (hPSCs), followed by the differentiation of these genetically engineered hPSCs into forebrain organoids. The resulting ChR2‐expressing human forebrain organoids showed homogeneous cellular expression of ChR2 throughout entire regions without any structural and functional perturbations and displayed consistent and robust neural activation upon light stimulation, allowing for the non‐virus mediated, spatiotemporal optogenetic control of neural activities. Furthermore, in the hybrid platform in which brain organoids are connected with spinal cord organoids and skeletal muscle spheroids, ChR2 knocked‐in forebrain organoids induced strong and consistent muscle contraction upon brain‐specific optogenetic stimulation. Our study thus provides a novel, non‐virus mediated, preclinical human organoid system for light‐inducible, consistent control of neural activities to study neural circuits and dynamics in normal and disease‐specific human brains as well as neural connections between brain and other peripheral tissues. [ABSTRACT FROM AUTHOR]

Wound healing in adults largely depends on the functional state of multipotent mesenchymal stromal cells (MSCs). Human fetal tissues at the early stages of development are known to heal quickly with a full-quality restoration of the original structure. The differences in the molecular mechanisms that determine the functional activity of mesodermal cells in fetuses and adults remain virtually unknown. Using two independent human induced pluripotent stem cell (iPSC) lines, we examined the effects of the initial WNT and BMP activation on the differentiation of iPSCs via mesodermal progenitors into MSCs and highlighted the functions of these cells that are altered by the proinflammatory microenvironment. The WNT-induced mesoderm commitment of the iPSCs enhanced the expression of paraxial mesoderm (PM)-specific markers, while the BMP4-primed iPSCs exhibited increased levels of lateral mesoderm (LM)-specific genes. The inflammatory status and migration rate of the isogenic iPSC-derived mesoderm cells were assessed via gene expression analysis and scratch assay under the receptor-dependent activation of the proinflammatory IFN-γ or TNF-α signaling pathway. Reduced IDO1 and ICAM1 expression levels were detected in the WNT- and BMP-induced MSC progenitors compared to the isogenic MSCs in response to stimulation with IFN-γ and TNF-α. The WNT- and BMP-induced MSC progenitors exhibited a higher migration rate than isogenic MSCs upon IFN-γ exposure. The established isogenic cellular model will provide new opportunities to elucidate the mechanisms of regeneration and novel therapeutics for wound healing. [ABSTRACT FROM AUTHOR]

Approval of induced pluripotent stem cells (iPSCs) for the manufacture of cell therapies to support clinical trials is now becoming realized after 20 years of research and development. In 2022 the International Society for Cell and Gene Therapy (ISCT) established a Working Group on Emerging Regenerative Medicine Technologies, an area in which iPSCs-derived technologies are expected to play a key role. In this article, the Working Group surveys the steps that an end user should consider when generating iPSCs that are stable, well-characterised, pluripotent, and suitable for making differentiated cell types for allogeneic or autologous cell therapies. The objective is to provide the reader with a holistic view of how to achieve high-quality iPSCs from selection of the starting material through to cell banking. Key considerations include: (i) intellectual property licenses; (ii) selection of the raw materials and cell sources for creating iPSC intermediates and master cell banks; (iii) regulatory considerations for reprogramming methods; (iv) options for expansion in 2D vs. 3D cultures; and (v) available technologies and equipment for harvesting, washing, concentration, filling, cryopreservation, and storage. Some key process limitations are highlighted to help drive further improvement and innovation, and includes recommendations to close and automate current open and manual processes. [ABSTRACT FROM AUTHOR]

With the continuous development and advancement of human pluripotent stem cell (PSC)-derived cell therapies, an ever-increasing number of clinical indications can benefit from their application. Due to the capacity for PSCs to form teratomas, safety testing is required to ensure the absence of residual PSCs in a cell product. To mitigate these limitations, in vitro analytical methods can be utilized as quality control after the production of a PSC-derived cell product. Sensitivity of these analytic methods is critical in accurately quantifying residual PSC in the final cell product. In this study, we compared the sensitivity of three in vitro assays: qPCR, ddPCR and RT-LAMP. The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A. The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts. In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy. [ABSTRACT FROM AUTHOR]

In this protocol, we detail a seven-stage differentiation methodology for generating pancreatic delta cells (SC-delta cells) from human pluripotent stem cells (hPSCs). In the first step, definitive endoderm is generated by activin A and CHIR99021, followed by induction of primitive gut tube and posterior foregut by treatment with FGF7, SANT1, LDN193189, PdBU, and retinoic acid (RA). The subsequent endocrine generation and directed SC-delta cell induction is achieved by a combined treatment of the FGF7 with FGF2 during stage 4 and 5, together with RA, XXI, ALK5 inhibitor II, SANT1, Betacellulin and LDN193189. The planar cultivation is converted to a suspended system after stage 5, allowing cells to aggregate into delta cell-containing spheroids. The differentiation takes approximately 4-5 weeks for delta cell generation and an additional 1-2 weeks for cell expansion and evaluation. We believe that this amenable and simplified protocol can provide a stable source of SC-delta cells from efficient differentiation, facilitating further investigation of the physiological role of delta cells as well as refinement of islet cell therapeutic strategies. [ABSTRACT FROM AUTHOR]

Introduction: Neurogenesis is tightly regulated in space and time, ensuring the correct development and organization of the central nervous system. Critical regulators of brain development and morphogenesis in mice include two members of the p53 family: p53 and p73. However, dissecting the in vivo functions of these factors and their various isoforms in brain development is challenging due to their pleiotropic effects. Understanding their role, particularly in neurogenesis and brain morphogenesis, requires innovative experimental approaches. Methods: To address these challenges, we developed an efficient and highly reproducible protocol to generate mouse brain organoids from pluripotent stem cells. These organoids contain neural progenitors and neurons that self-organize into rosette-like structures resembling the ventricular zone of the embryonic forebrain. Using this model, we generated organoids from p73-deficient mouse cells to investigate the roles of p73 and its isoforms (TA and DNp73) during brain development. Results and Discussion: Organoids derived from p73-deficient cells exhibited increased neuronal apoptosis and reduced neural progenitor proliferation, linked to compensatory activation of p53. This closely mirrors previous in vivo observations, confirming that p73 plays a pivotal role in brain development. Further dissection of p73 isoforms function revealed a dual role of p73 in regulating brain morphogenesis, whereby TAp73 controls transcriptional programs essential for the establishment of the neurogenic niche structure, while DNp73 is responsible for the precise and timely regulation of neural cell fate. These findings highlight the distinct roles of p73 isoforms in maintaining the balance of neural progenitor cell biology, providing a new understanding of how p73 regulates brain morphogenesis. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects not only the joints but also has significant cardiovascular (CV) manifestations. The mechanistic interplay between RA and cardiovascular complications is not yet well understood due to the lack of relevant in vitro models. In this study, we established RA cardiac microtisses (cMTs) from iPSC-derived cardiomyocytes (CMs), endothelial cells (ECs) and cardiac fibroblasts (CFs) to investigate whether this fully human 3D multicellular system could serve as a platform to elucidate the connection between RA and CV disorders. Methods: PBMC and FLS from healthy and RA donors were reprogrammed to hiPSCs with Sendai vectors. hiPSCs pluripotency was assessed by IF, FACS, spontaneous embryoid bodies formation and teratoma assay. hiPSCs were differentiated to cardiac derivatives such as CMs, ECs and CFs, followed by cell markers characterizations (IF, FACS, qRT-PCR) and functional assessments. 3D cMTs were generated by aggregation of 70% CMs, 15% ECs and 15% CFs. After 21 days in culture, structural and metabolic properties of 3D cMTs were examined by IF, qRT-PCR and Seahorse bioanalyzer. Results: hiPSCs demonstrated typical colony-like morphology, normal karyotype, presence of pluripotency markers, and ability to differentiate into cells originating from all three germ layers. hiPSC-CMs showed spontaneous beating and expression of cardiac markers (cTnT, MYL7, NKX2.5, MYH7). hiPSC-ECs formed sprouting spheres and tubes and expressed CD31 and CD144. hiPSC-CFs presented spindle-shaped morphology and expression of vimentin, collagen 1 and DDR2. Self-aggregation of CMs/ECs/CFs allowed development of contracting 3D cMTs, demonstrating spherical organization of the cells, which partially resembled the cardiac muscle, both in structure and function. IF analysis confirmed the expression of cTnT, CD31, CD144 and DDR2 in generated 3D cMTs. RA cMTs exhibited significantly greater formation of capillary-like structures, mimicking enhanced vascularization—key RA feature—compared to control cMTs. Seahorse examination of cMTs revealed changes in mitochondrial and glycolytic rates in the presence of metabolic substrates and inhibitors. Conclusions: The cMTs model may represent an advanced human stem cell-based platform for modeling CV complications in RA. The highly developed capillary-like structures observed within RA cMTs highlight a critical feature of inflammation-induced CV dysfunction in chronic inflammatory diseases. [ABSTRACT FROM AUTHOR]

The extracellular matrix plays a critical role in modulating cell behaviour in the developing and adult central nervous system influencing neural cell morphology, function and growth. Neurons and astrocytes, play vital roles in neural signalling and support respectively and respond to cues from the surrounding matrix environment. However, a better understanding of the impact of specific individual extracellular matrix proteins on both neurons and astrocytes is critical for advancing the development of matrix‐based scaffolds for neural repair applications. This study aimed to provide an in‐depth analysis of how different commonly used extracellular matrix proteins— laminin‐1, Fn, collagen IV, and collagen I—affect the morphology and growth of trophic induced pluripotent stem cell (iPSC)‐derived astrocyte progenitors and mouse motor neuron‐like cells. Following a 7‐day culture period, morphological assessments revealed that laminin‐1, fibronectin, and collagen‐IV, but not collagen I, promoted increased process extension and a stellate morphology in astrocytes, with collagen‐IV yielding the greatest increases. Subsequent analysis of neurons grown on the different extracellular matrix proteins revealed a similar pattern with laminin‐1, fibronectin, and collagen‐IV supporting robust neurite outgrowth. fibronectin promoted the greatest increase in neurite extension, while collagen‐I did not enhance neurite growth compared to poly‐L‐lysine controls. Super‐resolution microscopy highlighted extracellular matrix‐specific nanoscale changes in cytoskeletal organization, with distinct patterns of actin filament distribution where the three basement membrane‐associated proteins (laminin‐1, fibronectin, and collagen‐IV) promoted the extension of fine cellular processes. Overall, this study demonstrates the potent effect of laminin‐1, fibronectin and collagen‐IV to promote both iPSC‐derived astrocyte progenitor and neuronal growth, yielding detailed insights into the effect of extracellular matrix proteins on neural cell morphology at both the whole cell and nanoscale levels. The ability of laminin‐1, collagen‐IV and fibronectin to elicit strong growth‐promoting effects highlight their suitability as optimal extracellular matrix proteins to incorporate into neurotrophic biomaterial scaffolds for the delivery of cell cargoes for neural repair. [ABSTRACT FROM AUTHOR]

Background: Atrial fibrillation (AF) stands as a prevalent and detrimental arrhythmic disorder, characterized by intricate pathophysiological mechanisms. The availability of reliable and reproducible AF models is pivotal in unraveling the underlying mechanisms of this complex condition. Unfortunately, the researchers are still confronted with the absence of consistent in vitro AF models, hindering progress in this crucial area of research. Methods: Human induced pluripotent stem cells derived atrial myocytes (hiPSC-AMs) were generated based on the GiWi methods and were verified by whole-cell patch clamp, immunofluorescent staining, and flow cytometry. Then hiPSC-AMs were employed to establish the AF model by HS. Whole-cell patch clamp technique and calcium imaging were used to identify the AF model. The stability of 29 reference genes was evaluated using delta-Ct, GeNorm, NormFinder, and BestKeeper algorithms; Results: HiPSC-AMs displayed atrial myocyte action potentials and expressed the atrial-specific protein MLC-2 A and NR2F2, about 70% of the cardiomyocytes were MLC-2 A positive. After HS, hiPSC-AMs showed a significant increase in beating frequency, a shortened action potential duration, and increased calcium transient frequency. Of the 29 candidate genes, the top five most stably ranked genes were ABL1, RPL37A, POP4, RPL30, and EIF2B1. After normalization using ABL1, KCNJ2 was significantly upregulated in the AF model; Conclusions: In the hiPSC-AMs AF model established by HS, ABL1 provides greater normalization efficiency than commonly used GAPDH. [ABSTRACT FROM AUTHOR]

Nutrient-sensing pathways undergo deregulation in aged animals, exerting a pivotal role in regulating the cell cycle and subsequent stem cell division. Nevertheless, their precise functions in governing pluripotent stem cell differentiation remain largely elusive. Here, we uncovered a significant alteration in the cellular constituents of the intestinal epithelium in aged humans and mice. Employing Drosophila midgut and mouse organoid culture models, we made an observation regarding the altered trajectory of differentiation in intestinal stem cells (ISC) during overnutrition or aging, which stems from the erroneous activation of the insulin receptor signaling pathway. Through genetic analyses, we ascertained that the nutrient-sensing pathway regulated the direction of ISC differentiation by modulating the maturation of endosomes and SOX21A transcription factor. This study elucidates a nutrient-sensing pathway-mediated mechanism underlying stem cell differentiation, offering insights into the etiology of stem cell dysfunction in aged animals, including humans. Nutrient-sensing pathways undergo deregulation in aged animals. Here the authors uncover a significant alteration in the composition of intestinal epithelial cells during aging which is caused by changes to the differentiation direction of intestinal stem cells. [ABSTRACT FROM AUTHOR]

Introduction: The changing composition of non-cell autonomous circulating factors in blood as humans age is believed to play a role in muscle mass and strength loss. The mechanisms through which these circulating factors act in age-related skeletal muscle changes is not fully understood. In this study, we used human myogenic progenitors derived from human pluripotent stem cells to study non-cell autonomous roles of circulating factors during the process of myogenic differentiation. Methods: Myogenic progenitors from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) were supplemented with serum samples fromaged or young Fischer 344 × Brown Norway F1-hybrid rats. The effect of aged or young serum supplementation on myogenic progenitor proliferation, myotube formation capacity, differentiation, and early transcriptomic profiles were analyzed. Results: We found that aged rat serum supplementation significantly reduced cell proliferation and increased cell death in both ESC- and iPSC-derived myogenic progenitors. Next, we found that the supplementation of aged rat serum inhibited myotube formation and maturation during terminal differentiation from progenitors to skeletal myocytes when compared to the cells treated with young adult rat serum. Lastly, we identified that gene expression profiles were affected following serum supplementation in culture. Discussion: Together, aged serum supplementation caused cellular and transcriptomic changes in human myogenic progenitors. The current data from our in vitro model possibly simulate non-cell autonomous contributions of blood composition to age-related processes in human skeletal muscle. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells (HSCs) obtained from patient-derived human induced pluripotent stem cells (iPSCs) are a promising tool for curing various hematological disorders. We previously demonstrated that enforced expression of the LIM-homeobox transcription factor Lhx2, which is essential for mouse embryonic hematopoiesis, leads to generation of engraftable and expandable hematopoietic stem cells (HSCs) from mouse iPSCs. However, it remained unknown whether Lhx2 can induce HSCs from human iPSCs. Here, we investigated the effect of Lhx2 overexpression on hematopoietic differentiation of human iPSCs. Unexpectedly, Lhx2 severely inhibited proliferation of human iPSC-derived hematopoietic cells. Thus, Lhx2 exhibited differential effects on mouse and human hematopoietic cells. Further studies implied that the inhibitory effect of Lhx2 on human iPSC-derived hematopoietic cells was due to insufficient transcriptional activation ability. Therefore, we modified Lhx2 to strengthen its activity as a transcriptional activator. This modified Lhx2 could induce ex vivo amplification of human iPSC-derived hematopoietic stem/progenitor cells (HSPCs). We believe that these findings will facilitate the development of a method to efficiently produce HSCs from human iPSCs. [ABSTRACT FROM AUTHOR]

Spinal cord injury creates an inflammatory microenvironment that regulates the capacity of transplanted human Neural Stem Cells (hNSC) to migrate, differentiate, and repair injury. Despite similarities in gene expression and markers detected by immunostaining, hNSC populations exhibit heterogeneous therapeutic potential. This heterogeneity derives in part from the epigenetic landscape in the hNSC genome, specifically methylation (5mC) and hydroxymethylation (5hmC) state, which may affect the response of transplanted hNSC in the injury microenvironment and thereby modulate repair capacity. We demonstrate a significant up-regulation of methylcytosine dioxygenase 2 gene (TET2) expression in undifferentiated hNSC derived from human embryonic stem cells (hES-NSC), and report that this is associated with hES-NSC competence for differentiation marker expression. TET2 protein catalyzes active demethylation and TET2 upregulation could be a signature of pluripotent exit, while shaping the epigenetic landscape in hES-NSC. We determine that the inflammatory environment overrides epigenetic programming in vitro and in vivo by directly modulating TET2 expression levels in hES-NSC to change cell fate. We also report the effect of cell fate and microenvironment on differential methylation 5mC/5hmC balance. Understanding how the activity of epigenetic modifiers changes within the transplantation niche in vivo is crucial for assessment of hES-NSC behavior for potential clinical applications. [ABSTRACT FROM AUTHOR]

The Cornelia de Lange syndrome (CdLS) is a rare genetic disease, which is characterized by a cohesinopathy. Mutations of the NIPBL gene are observed in 65% of CdLS patients. A novel iPSC (induced Pluripotent Stem Cell) line was reprogrammed from the leukocytes of a CdLS patient carrying a missense mutation of the NIPBL gene. A mutation-corrected isogenic iPSC-line and two iPSC-lines generated from the healthy parents were used as controls. The iPSC lines were differentiated along the hepatocyte-lineage. Comparative immunofluorescence, RNA-seq and ATAC-seq analyses were performed on undifferentiated and differentiated iPSCs. In addition, chromatin organization was studied by ChIP-Seq analysis on the patient derived iPSCs as well as the respective controls. Relative to the mutation-corrected and the healthy-parents iPSCs, the patient-derived counterparts are defective in terms of differentiation along the hepatocyte-lineage. One-third of the genes selectively up-regulated in CdLS-derived iPSCs and hepatic cells are non-protein-coding genes. By converse, most of the selectively down-regulated genes code for transcription factors and proteins regulating neural differentiation. Some of the transcriptionally silenced loci, such as the DPP6 gene on chromosome 7q36.2 and the ZNF gene cluster on chromosome 19p12, are located in closed-chromatin regions. Relative to the corresponding controls, the global transcriptomic differences observed in CdLS undifferentiated iPSCs are associated with altered chromatin accessibility, which was confirmed by ChIP-Seq analysis. Thus, the deficits in the differentiation along the hepatocyte lineage observed in our CdLS patient is likely to be due to a transcriptional dysregulation resulting from a cohesin-dependent alteration of chromatin accessibility. [ABSTRACT FROM AUTHOR]

Background: Spinal cord injury (SCI) is a severe traumatic spinal condition with a poor prognosis. In this study, a scaffold called linearly ordered collagen aggregates (LOCAS) was created and loaded with induced pluripotent stem cells (iPSCs)-derived neural stem cells (NSCs) from human umbilical cord blood derived mesenchymal stem cells (hUCB-MSCs) to treat SCI in a rat model. Methods: The rats underwent a complete transection SCI resulting in a 3-mm break at either the T9 or T10 level of the spinal cord. Results: Scanning electron microscope analysis revealed a uniform pore structure on the coronal plane of the scaffold. The LOCAS had a porosity of 88.52% and a water absorption of 1161.67%. Its compressive modulus and stress were measured at 4.1 MPa and 205 kPa, respectively, with a degradation time of 10 weeks. After 12 weeks, rats in the LOCAS-iPSCs-NSCs group exhibited significantly higher BBB scores (8.6) compared to the LOCAS-iPSCs-NSCs group (5.6) and the Model group (4.2). The CatWalk analysis showed improved motion trajectory, regularity index (RI), and swing speed in the LOCAS-iPSCs-NSCs group compared to the other groups. Motor evoked potentials latency was lower and amplitude was higher in the LOCAS-iPSCs-NSCs group, indicating better neural function recovery. Histological analysis demonstrated enhanced neuronal differentiation of NSCs and nerve fiber regeneration promoted by LOCAS-iPSCs-NSCs, leading to improved motor function recovery in rats. The LOCAS scaffold facilitated ordered neurofilament extension and guided nerve regeneration. Conclusions: The combination of LOCAS and iPSCs-NSCs demonstrated a positive therapeutic impact on motor function recovery and tissue repair in rats with SCI. This development offers a more resilient bionic microenvironment and presents novel possibilities for clinical SCI repair. [ABSTRACT FROM AUTHOR]

Background: Islet transplantation is a recommended treatment for type 1 diabetes but is limited by donor organ shortage. This study introduces an innovative approach for improving the differentiation and functionality of insulin-producing cells (IPCs) from iPSCs using 3D spheroid formation and hydrogel matrix as an alternative pancreatic islet source. The extracellular matrix (ECM) is crucial for pancreatic islet functionality, but finding the ideal matrix for β-cell differentiation has been challenging. We aimed to advance IPC differentiation and maturation through an esterified collagen hydrogel, comparing its effectiveness with conventional basement membrane extract (BME) hydrogels. Methods: iPSCs were differentiated into IPCs using a small molecule-based sequential protocol, followed by spheroid formation in concave microwells. Rheological analysis, scanning electron microscopy, and proteomic profiling were used to characterize the chemical and physical properties of each matrix. IPCs, both in single-cell form and as spheroids, were embedded in either ionized collagen or BME hydrogels, which was followed by assessments of morphological changes, pancreatic islet-related gene expression, insulin secretion, and pathway activation using comprehensive analytical techniques. Results: Esterified collagen hydrogels markedly improved the structural integrity, insulin expression, and cell-cell interactions in IPC spheroids, forming densely packed insulin-expressing clusters, in contrast to the dispersed cells observed in BME cultures. Collagen hydrogel significantly enhanced the mRNA expression of crucial endocrine markers and maturation factors, with IPC spheroids showing accelerated differentiation from day 5, suggesting a faster differentiation compared to single cells in hydrogel encapsulation. Insulin secretion in response to glucose in collagen environments, with a GSIS index of 2.46 ± 0.05, exceeded those in 2D and BME, demonstrating superior pancreatic islet functionality. Pathway analysis highlighted enhanced insulin secretion capabilities, evidenced by the upregulation of genes like Secretogranin III and Chromogranin A in collagen cultures. In vivo transplantation results showed that collagen hydrogel enhanced cluster integrity, tissue integration, and insulin secretion compared to non-embedded IPCs and BME groups. Conclusion: Esterified collagen hydrogels demonstrated superior efficacy over 2D and BME in promoting IPC differentiation and maturation, possibly through upregulation of the expression of key secretion pathway genes. Our findings suggest that using collagen hydrogels presents a promising approach to enhance insulin secretion efficiency in differentiating pancreatic β-cells, advancing cell therapy in diabetes cell therapy. [ABSTRACT FROM AUTHOR]

Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM), in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome, qRT-PCR, Metascape, and KEGG pathway analyses were conducted to assess elevated proteins, genes, and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-γ), interleukin (IL)1-beta (IL-1β), IL-8 and IL-16. Enrichment analysis revealed malaria, positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria, MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally, HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ, activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore, our model is an alternative to rodent experimental models of CM. [ABSTRACT FROM AUTHOR]

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]

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]

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]