Many neurological diseases involving tissue damage cannot be treated with drug-based approaches, and the inaccessibility of human brain samples further hampers the study of these diseases. Human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provide an excellent model for studying neural development and function. PSCs can be differentiated into various neural cell types, providing a renewal source of functional human brain cells. Therefore, PSC-derived neural cells are increasingly used for multiple applications, including neurodevelopmental and neurotoxicological studies, neurological disease modeling, drug screening, and regenerative medicine. In addition, the neural cells generated from patient iPSCs can be used to study patient-specific disease signatures and progression. With the recent advances in genome editing technologies, it is possible to remove the disease-related mutations in the patient iPSCs to generate corrected iPSCs. The corrected iPSCs can differentiate into neural cells with normal physiological functions, which can be used for autologous transplantation. This review highlights the current progress in using PSCs to understand the fundamental principles of human neurodevelopment and dissect the molecular mechanisms of neurological diseases. This knowledge can be applied to develop better drugs and explore cell therapy options. We also discuss the basic requirements for developing cell transplantation therapies for neurological disorders and the current status of the ongoing clinical trials.

The piggyBac+TET-on transposon induction system has a high efficiency in integrating exogenous genes in multiple cell types, can precisely integrate to reduce genomic damage, has a flexible gene expression regulation, and a strong genetic stability. When used in conjunction with somatic cell nuclear transfer experiments, it can precisely and effectively reveal the intrinsic mechanisms of early biological development. This study successfully reprogrammed black-boned sheep fibroblasts (SFs) into induced pluripotent stem cells (iPSCs) using the piggyBac+TET-on transposon system and investigated their impact on early embryonic development. Seven exogenous reprogramming factors (bovine OCT4, SOX2, KLF4, cMyc, porcine NANOG, Lin-28, and SV40 Large T) were delivered into SFs, successfully inducing iPSCs. A growth performance analysis revealed that iPSC clones exhibited a raised or flat morphology with clear edges, positive alkaline phosphatase staining, and normal karyotypes. The transcriptome analysis indicated a significant enrichment of iPSCs in oxidative phosphorylation and cell proliferation pathways, with an up-regulated expression of the ATP5B, SDHB, Bcl-2, CDK1, and Cyclin D1 genes and a down-regulated expression of BAX (p < 0.05). Somatic cell nuclear transfer experiments demonstrated that the cleavage rate (85% ± 2.12) and blastocyst rate (52% ± 2.11) of the iPSCs were significantly higher than those of the SFs (p < 0.05). The detection of trilineage marker genes confirmed that the expression levels of endoderm (DCN, NANOS3, FOXA2, FOXD3, SOX17), mesoderm (KDR, CD34, NFH), and ectoderm (NEUROD) markers in iPSCs were significantly higher than in SFs (p < 0.01). The findings demonstrate that black-boned sheep iPSCs possess pluripotency and the potential to differentiate into all three germ layers, revealing the mechanisms by which reprogrammed iPSCs influence early embryonic development and providing a critical foundation for research on sheep pluripotent stem cells.

Over the past two decades, significant advancements have been made in the induced pluripotent stem cell (iPSC) technology. These developments have enabled the broader application of iPSCs in neuroscience, improved our understanding of disease pathogenesis, and advanced the investigation of therapeutic targets and methods. Specifically, optimizations in reprogramming protocols, coupled with improved neuronal differentiation and maturation techniques, have greatly facilitated the generation of iPSC-derived neural cells. The integration of the cerebral organoid technology and CRISPR/Cas9 genome editing has further propelled the application of iPSCs in neurodegenerative diseases to a new stage. Patient-derived or CRISPR-edited cerebral neurons and organoids now serve as ideal disease models, contributing to our understanding of disease pathophysiology and identifying novel therapeutic targets and candidates. In this review, we examine the development of iPSC-based models in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease.

The repair of peripheral nerve injury (PNI) presents a multifaceted and protracted challenge, with current therapeutic approaches failing to achieve optimal repair outcomes, thereby not satisfying the considerable clinical demand. The advent of tissue engineering has led to a growing body of experimental evidence indicating that the synergistic application of nerve conduits, which provide structural guidance, alongside the biological signals derived from exosomes and stem cells, yields superior therapeutic results for PNI compared to isolated interventions. This combined approach holds great promise for clinical application. In this review, we present the latest advancements in the treatment of PNI through the integration of stem cells or exosomes with nerve conduits. We have addressed the inadequate efficiency of exosomes or stem cells in conjunction with nerve conduits from 3 perspectives: enhancing stem cells or exosomes, improving nerve conduits, and incorporating physical stimulation.

Epithelial-mesenchymal transition (EMT) is closely linked to liver cancer prognosis, invasiveness, and aggressiveness. One promising treatment for liver cancer is cell therapy, where stem cells are stimulated to develop into functional liver cells. This study aimed to investigate the effect of miR-122-5p on the differentiation of human induced pluripotent stem cells (hiPSCs) into hepatocyte-like cells and its impact on the EMT process in liver cancer cells. MiR-122-5p was overexpressed or silenced in hiPSCs to analyze the expression of liver-specific markers, including AFP, ALB and ASGPR, to confirm hepatocyte-like differentiation. A co-culture system with HepG2 liver cancer cells was also used to evaluate the effect of miR-122-5p-overexpressing hiPSCs or miR-122-5p-silencing hiPSCs on the expression of EMT markers. Results revealed that overexpression of miR-122-5p in hiPSCs induced hepatocyte-like characteristics, as evidenced by increased levels of AFP, ALB, and ASGPR. However, knockdown of miR-122-5p had the opposite effect. In the co-culture system, hiPSCs overexpressing miR-122-5p inhibited the EMT process of HepG2 cells, resulting in increased levels of mesenchymal markers and decreased levels of epithelial markers. Taken together, miR-122-5p promotes the differentiation of hiPSCs into hepatocyte-like cells and inhibits EMT process of liver cancer cells. Targeting miR-122-5p may be a novel approach to prevent liver cancer progression through cell therapy.

Diabetic nephropathy (DN) is a prevalent complication of diabetes, with current treatment options offering limited effectiveness, particularly in advanced stages. Human pluripotent stem cells (hPSCs), particularly induced PSCs (iPSCs), show promising potential in the treatment of DN due to their pluripotency, capacity for differentiation into kidney-specific cells, and suitability for personalized therapies. iPSC-based personalized approaches can effectively mitigate immune rejection, a common challenge with allogeneic transplants, thus enhancing therapeutic outcomes. Clustered regularly interspaced short palindromic repeats (CRISPR) gene editing further enhances the potential of hPSCs by enabling the precise correction of disease-associated genetic defects, increasing both the safety and efficacy of therapeutic cells. In addition to direct treatment, hPSCs have proven valuable in disease modeling and drug screening, particularly for identifying and validating disease-specific targets. Kidney organoids derived from hPSCs replicate key features of DN pathology, making them useful platforms for validating therapeutic targets and assessing drug efficacy. Comparatively, both hPSCs and mesenchymal SCs (MSCs) have shown promise in improving renal function in preclinical models, with hPSCs offering broader differentiation capacity. Integration with tissue engineering technologies, such as three-dimensional bioprinting and bioengineered scaffolds, expands the regenerative potential of hPSCs by supporting the formation of functional renal structures and enhancing in vivo integration and regenerative capacity. Despite current challenges, such as tumorigenicity, genomic instability, and limited direct research, advances in gene editing, differentiation protocols, and tissue engineering promise to address these barriers. Continued optimization of these approaches will likely lead to successful clinical applications of hPSCs, potentially revolutionizing treatment options for DN.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common renal genetic disease, with most patients carrying mutations in PKD1. The main feature is the formation of bilateral renal cysts, leading to end stage renal failure in a significant proportion of those affected. Despite recent advances made in understanding ADPKD, there are currently no effective curative therapies. The emergence of human induced pluripotent stem cell (hiPSC)-derived kidney disease models has led to renewed hope that more physiological systems will allow for the development of novel treatments. hiPSC-derived organoid models have been used to recapitulate ADPKD, however they present numerous limitations which remain to be addressed. In the present study, we report an efficient method for generating organoids containing a network of polarised and ciliated epithelial tubules. PKD1 null (PKD1-/-) organoids spontaneously develop dilated tubules, recapitulating early ADPKD cystogenesis. Furthermore, PKD1-/- tubules present primary cilia defects when dilated. Our model could therefore serve as a valuable tool to study early ADPKD cystogenesis and to develop novel therapies.

Pluripotent stem cells provide opportunities for treating injuries and previously incurable diseases. A major concern is the immunogenicity of stem cells and their progeny. Here, we have dissected the molecular mechanisms that allow natural killer (NK) cells to respond to human pluripotent stem cells, investigating a wide selection of activating and inhibitory NK-cell receptors and their ligands. Reporter cells expressing the activating receptor NKG2D responded strongly to embryonic stem (ES) cell lines and induced pluripotent stem (iPS) cell lines, whereas reporter cells expressing the activating receptors NKp30, NKp46, KIR2DS1, KIR2DS2, and KIR2DS4 did not respond. Human ES and iPS cells invariably expressed several ligands for NKG2D. Expression of HLA-C and HLA-E was lacking or low, insufficient to trigger reporter cells expressing the inhibitory receptors KIR2DL1, -2DL2, or -2DL3. Similar results were obtained for the pluripotent embryonic carcinoma cell lines NTERA-2 and 2102Ep, and also iPS-cell-derived neural progenitor cells. Importantly, neural progenitor cells and iPS-cell-derived motoneurons also expressed B7H6, the ligand for the activating receptor NKp30. In line with these observations, IL-2-stimulated NK cells showed robust cytotoxic responses to ES and iPS cells as well as to iPS-cell-derived motoneurons. No significant differences in cytotoxicity levels were observed between KIR/HLA matched and mismatched combinations of NK cells and pluripotent targets. Together, these data indicate that pluripotent stem cells and their neural progeny are targets for NK-cell killing both by failing to sufficiently express ligands for inhibitory receptors and by expression of ligands for activating receptors.

Cardiovascular diseases remain one of the leading causes of death worldwide. Given the limited self-repair capacity of cardiac tissue, cardiac tissue engineering (CTE) aims to develop strategies and materials for repairing or replacing damaged cardiac tissue by combining biology, medicine, and engineering. Indeed, CTE has made significant strides since the discovery of induced pluripotent stem cells (iPSCs) in 2006, including creating cardiac patches, organoids, and chip models derived from iPSCs, thus offering new strategies for treating cardiac diseases.

A systematic search for relevant literature published between 2003 and 2024 was conducted in the PubMed and Web of Science databases using "Cardiac Tissue Engineering", "3D Bioprinting", "Scaffold in Tissue Engineering", "Induced Pluripotent Stem Cells", and "iPSCs" as keywords.

This systematic search using the abovementioned keywords identified relevant articles for inclusion in this review. The resulting literature indicated that CTE can offer innovative solutions for treating cardiac diseases when integrated with three-dimensional (3D) bioprinting and iPSC technology.

Despite notable advances in the field of CTE, multiple challenges remain relating to 3D-bioprinted cardiac tissues. These include maintaining long-term cell viability, achieving precise cell distribution, tissue vascularization, material selection, and cost-effectiveness. Therefore, further research is needed to optimize printing techniques, develop more advanced bio-inks, explore larger-scale tissue constructs, and ensure the biosafety and functional fidelity of engineered cardiac tissues. Subsequently, future research efforts should focus on these areas to facilitate the clinical translation of CTE. Moreover, additional long-term animal models and preclinical studies should be conducted to ensure the biosafety and functionality of engineered cardiac tissues, thereby creating novel possibilities for treating patients with heart diseases.

The therapeutic armamentarium for heart failure with preserved ejection fraction (HFpEF) remains notably constrained. A factor contributing to this problem could be the scarcity of in vitro models for HFpEF, which hinders progress in developing new therapeutic strategies. Here, we aimed at developing a novel, comorbidity-inspired, human, in vitro model for HFpEF.

Human induced pluripotent stem cells-derived cardiomyocytes were used to produce cardiac organoids. The generated organoids were then subjected to HFpEF-associated, comorbidity-inspired conditions, such as hypertension, diabetes, and obesity-related inflammation. To assess the development of HFpEF pathophysiological features, organoids were thoroughly evaluated for their structural, functional, electrophysiological, and metabolic properties.

Exposure to the combination of all comorbidity-mimicking conditions resulted in the largest cellular volume of 1692±52 versus 1346±84 µm3 in RPMI (Roswell Park Memorial Institute medium) control group (P=0.003), while lower in obesity, hypertension, and diabetes groups: 1059±40 µm3 (P=0.014), 1276±35 µm3 (P=0.940), and 1575±70 µm3 (P=0.146), respectively. Similarly, ultrastructural fibrosis was most significantly observed after exposure to the combination of all HFpEF-inducing conditions 14.6±1.2% compared with single condition exposure 5.2±1.3% (obesity), 6.7±3.5% (hypertension), and 9.0±1.1% (diabetes; P<0.001). Moreover, HFpEF-related conditions led to an increase in passive force compared with control (7.52±1.08 versus 2.33±0.46 mN/mm, P<0.001), whereas no significant alterations were noted in active contractile forces. Relaxation constant τ was significantly prolonged after exposure to HFpEF conditions showing a prolongation of 95.9 ms (36.4-106.4; P=0.028) compared with a shortening of 35.6 ms (43.3-67.3; P=0.80) in the control. Finally, organoid exposure to HFpEF conditions led to a significant increase in oxidative stress levels and a significant decline in oxygen consumption rate.

We established a novel, human, in vitro model for HFpEF, based on comorbidity-inspired conditions. The model faithfully recapitulated the structural, functional, and mechanistic features of HFpEF. This model holds the potential to provide mechanistic insights and facilitate the identification of novel therapeutic targets.

Stem cells play a critical role in the regeneration process by proliferating and differentiating to form new bone tissue. However, stem cells tend to lose their stemness and pluripotency during in vitro expansion, resulting in reduced bone regeneration capacity after osteogenic induction. Our aim is to enhance the osteogenic impact of human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) through spontaneous spheroid in vitro. The pluripotency and osteogenesis-related genes up-regulated in hUC-MSCs can be enhanced in spontaneous spheroids in vitro. For in vivo testing, spontaneous spheroids were transplanted into mice using beta-tricalcium phosphate as a scaffold. Transplant samples were stained using hematoxylin and eosin (HE), immunohistochemistry, and TRAP staining. The samples showed new bone formation, upregulated SP7 and OCN expression, and more vigorous bone metabolism in the Sph-OI group than the other groups. However, new bone formation was mainly immature bone. Overall, our findings demonstrate that hUC-MSC spheroids possess remarkable pluripotency, with the spontaneous spheroids formed following osteogenic induction exhibiting enhanced osteogenic differentiation potential and bone regeneration capacity. However, optimizing the osteogenic differentiation process and elucidating the underlying mechanisms of bone regeneration are critical scientific issues that urgently need to be addressed to enable its application in bone regeneration.

Cryptococcal meningitis (CM) is a significant global health issue, particularly affecting individuals with HIV. Amphotericin B (AmB) serves as the cornerstone treatment for CM; however, its clinical application is restricted due to limited penetration of the blood-brain barrier and associated nephrotoxicity.

This study investigates the use of exosomes derived from induced pluripotent stem cells (iPSC-Exos) as carriers for AmB in treating CM, aiming to enhance therapeutic efficacy and safety and reduce AmB toxicity.

Exosomes were extracted from iPSC culture supernatants using ultrafiltration and ultracentrifugation. Their morphology and size were analyzed using transmission electron microscopy (TEM) and nanoparticle flow cytometry (nFCM). Purity was confirmed by Western blotting for markers CD9, CD63, and TSG101. AmB was loaded into iPSC-Exos using a co-incubation method. The cytotoxicity of the iPSC-Exo/AmB complex was evaluated on HEK 293 T and RAW264.7 cells using the CCK-8 assay, while apoptosis was assessed using live/dead cell staining and flow cytometry. The hemolytic effects were tested using rabbit red blood cells. In a C57BL/6 J mouse model of cryptococcal infection, treatment groups (AmB, iPSC-Exo/AmB, and iPSC-Exo) were administered corresponding drugs, with blood and brain samples collected for analysis. The minimum inhibitory concentration (MIC) of iPSC-Exo/AmB and conventional AmB against Cryptococcus was determined.

The iPSC-Exo/AmB complex exhibited reduced cytotoxicity in vitro and decreased AmB-induced renal and hepatic toxicity in vivo. Its MIC against Cryptococcus was over eight times lower than conventional AmB, significantly reducing fungal burden in the mouse brain and lowering serum inflammatory factors.

The iPSC-Exo/AmB complex is a promising therapeutic strategy that enhances AmB efficacy while reducing toxicity, offering new hope for treating CM and other refractory fungal infections of the central nervous system.

JOURNAL/nrgr/04.03/01300535-202502000-00034/figure1/v/2024-05-28T214302Z/r/image-tiff Several studies have found that transplantation of neural progenitor cells (NPCs) promotes the survival of injured neurons. However, a poor integration rate and high risk of tumorigenicity after cell transplantation limits their clinical application. Small extracellular vesicles (sEVs) contain bioactive molecules for neuronal protection and regeneration. Previous studies have shown that stem/progenitor cell-derived sEVs can promote neuronal survival and recovery of neurological function in neurodegenerative eye diseases and other eye diseases. In this study, we intravitreally transplanted sEVs derived from human induced pluripotent stem cells (hiPSCs) and hiPSCs-differentiated NPCs (hiPSC-NPC) in a mouse model of optic nerve crush. Our results show that these intravitreally injected sEVs were ingested by retinal cells, especially those localized in the ganglion cell layer. Treatment with hiPSC-NPC-derived sEVs mitigated optic nerve crush-induced retinal ganglion cell degeneration, and regulated the retinal microenvironment by inhibiting excessive activation of microglia. Component analysis further revealed that hiPSC-NPC derived sEVs transported neuroprotective and anti-inflammatory miRNA cargos to target cells, which had protective effects on RGCs after optic nerve injury. These findings suggest that sEVs derived from hiPSC-NPC are a promising cell-free therapeutic strategy for optic neuropathy.

Background/Objectives: The dual forces of structured inquiry and serendipitous discovery have long shaped neuropsychiatric research, with groundbreaking treatments such as lithium and ketamine resulting from unexpected discoveries. However, relying on chance is becoming increasingly insufficient to address the rising prevalence of mental health disorders like depression and schizophrenia, which necessitate precise, innovative approaches. Emerging technologies like artificial intelligence, induced pluripotent stem cells, and multi-omics have the potential to transform this field by allowing for predictive, patient-specific interventions. Despite these advancements, traditional methodologies such as animal models and single-variable analyses continue to be used, frequently failing to capture the complexities of human neuropsychiatric conditions. Summary: This review critically evaluates the transition from serendipity to precision-based methodologies in neuropsychiatric research. It focuses on key innovations such as dynamic systems modeling and network-based approaches that use genetic, molecular, and environmental data to identify new therapeutic targets. Furthermore, it emphasizes the importance of interdisciplinary collaboration and human-specific models in overcoming the limitations of traditional approaches. Conclusions: We highlight precision psychiatry's transformative potential for revolutionizing mental health care. This paradigm shift, which combines cutting-edge technologies with systematic frameworks, promises increased diagnostic accuracy, reproducibility, and efficiency, paving the way for tailored treatments and better patient outcomes in neuropsychiatric care.

Mortality and morbidity from cardiovascular diseases are common worldwide. In order to improve survival and quality of life for this patient population, extensive efforts are being made to establish effective therapeutic modalities. New treatment options are needed, it seems. In addition to treating cardiovascular diseases, cell therapy is one of the most promising medical platforms. One of the most effective therapeutic approaches in this area is stem cell therapy. In stem cell biology, multipotent stem cells and pluripotent stem cells are divided into two types. There is evidence that stem cell therapy could be used as a therapeutic approach for cardiovascular diseases based on multiple lines of evidence. The effectiveness of stem cell therapies in humans has been studied in several clinical trials. In spite of the challenges associated with stem cell therapy, it appears that resolving them may lead to stem cells being used in cardiovascular disease patients. This may be an effective therapeutic approach. By mounting these stem cells on biological scaffolds, their effect can be enhanced.

Stem cell research holds huge promise for regenerative medicine and disease modeling, making the understanding and optimization of stem cell culture a critical aspect of advancing these therapeutic applications. This comprehensive review provides an in-depth overview of stem cell culture, including general information, contemporary techniques, encountered problems, and future perspectives. The article begins by explaining the fundamental characteristics of various stem cell types, elucidating the importance of proper culture conditions in maintaining pluripotency or lineage commitment. A detailed exploration of established culture techniques sheds light on the evolving landscape of stem cell culture methodologies. Common challenges such as genetic stability, heterogeneity, and differentiation efficiency are thoroughly discussed, with insights into cutting-edge strategies and technologies aimed at addressing these hurdles. Moreover, the article delves into the impact of substrate materials, culture media components, and biophysical cues on stem cell behavior, emphasizing the intricate interplay between the microenvironment and cell fate decisions. As stem cell research advances, ethical considerations and regulatory frameworks become increasingly important, prompting a critical examination of these aspects in the context of culture practices. Lastly, the article explores emerging perspectives, including the integration of artificial intelligence and machine learning in optimizing culture conditions, and the potential applications of stem cell-derived products in personalized medicine. This comprehensive overview aims to serve as a valuable resource for researchers and clinicians, fostering a deeper understanding of stem cell culture and its key role in advancing regenerative medicine and biomedical research.

Fabry disease is a multi-organ disease, caused by mutations in the GLA gene and leading to a progressive accumulation of glycosphingolipids due to enzymatic absence or malfunction of the encoded alpha-galactosidase A. Since pathomechanisms are not yet fully understood and available treatments are not efficient for all mutation types and tissues, further research is highly needed. This research involves many different model types, with significant effort towards the establishment of an in vivo model. However, these models did not replicate the variety of symptoms observed in patients. As an alternative strategy, patient-derived somatic cells as well as patient-independent cell lines were used to model specific aspects of the disease in vitro. Fabry disease patients present different phenotypes according to the mutation and the level of residual enzyme activity, pointing to the necessity of personalized disease modeling. With the advent of induced pluripotent stem cells, the derivation of a multitude of disease-affected cell types became possible, even in a patient-specific and mutation-specific manner. Only recently, three-dimensional Fabry disease models were established that even more closely resemble the native tissue of investigated organs and will bring research closer to the in vivo situation. This review provides an overview of human in vitro models and their achievements in unravelling the Fabry disease pathomechanism as well as in elucidating current and future treatment strategies.

We aimed to investigate the effect and potential mechanism of enhancing Neuregulin1 (NRG1)/v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 4 (ErbB4) expression on the differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes.

We utilized CRISPR-CAS9 technology to knock in ErbB4 and obtained a single-cell clone IPSN-AAVS1-CMV-ErbB4 (iPSCs-ErbB4). Subsequently, we induced the differentiation of iPSCs into cardiomyocytes and quantified the number of beating embryoid bodies. Furthermore, quantitative real-time PCR assessed the expression of cardiomyocyte markers, including ANP (atrial natriuretic peptide), Nkx2.5 (NK2 transcription factor related locus 5), and GATA4 (GATA binding protein 4). On the 14th day of differentiation, we observed the α-MHC (α-myosin heavy chain)-positive area using immunofluorescent staining and conducted western blotting to detect the expression of cTnT (cardiac troponin) protein and PI3K/Akt signaling pathway-related proteins. Additionally, we intervened the iPSCs-ErbB4 + NRG1 group with the PI3K/Akt inhibitor LY294002 and observed alterations in the expression of cardiomyocyte differentiation-related genes.

The number of beating embryoid bodies increased after promoting the expression of NRG1/ErbB4 compared to the iPSCs control group. Cardiomyocyte markers ANP, Nkx2.5, and GATA4 significantly increased on day 14 of differentiation, and the positive area of α-MHC was three times that of the iPSCs control group. Moreover, there was a marked increase in cTnT protein expression. However, there was no significant difference in cardiomyocyte differentiation between the iPSCs-ErbB4 group and the iPSCs control group. Akt phosphorylation was significantly increased in the iPSCs-ErbB4 + NRG1 group. LY294002 significantly reversed the enhancing effect of NRG1/ErbB4 overexpression on Akt phosphorylation as well as the increase in α-MHC and cTnT expression.

In conclusion, promoting the expression of NRG1/ErbB4 induced the differentiation of iPSC into cardiomyocytes, possibly through modulation of the PI3K/Akt signaling pathway.

Exosomes are a subset of Extracellular vesicles (EVs) released by most cells in the body and can play a significant role in the intercellular connection. Researchers today claim that exosomes secreted by induced pluripotent stem cells (iPSCs) alone can play the same role as direct cell transplantation and, unlike iPSCs, do not lead to tumorigenesis. As a result, iPSC-derived exosomes (iPSC-Exos) have many applications in cell-free treatments and therapeutic effects on various diseases. Male infertility due to a defect or deficiency of spermatogonia to maintain spermatogenesis is one of the diseases that iPSC-Exos seems to be a new way to cure. However, the studies on the effect of iPSC-Exos on male infertility are very limited. In this review, we intend to provide a broader perspective on understanding the mechanisms of iPSC-Exos on spermatogenesis by collecting and reviewing some of the research conducted in this field.

Stem cells-derived extracellular vesicles (SC-EVs) have emerged as promising therapeutic agents for wound repair, recapitulating the biological effects of parent cells while mitigating immunogenic and tumorigenic risks. These EVs orchestrate wound healing processes, notably through modulating angiogenesis-a critical event in tissue revascularization and regeneration. This study provides a comprehensive overview of the multifaceted mechanisms underpinning the pro-angiogenic capacity of EVs from various stem cell sources within the wound microenvironment. By elucidating the molecular intricacies governing their angiogenic prowess, we aim to unravel the mechanistic repertoire underlying their remarkable potential to accelerate wound healing. Additionally, methods to enhance the angiogenic effects of SC-EVs, current limitations, and future perspectives are highlighted, emphasizing the significant potential of this rapidly advancing field in revolutionizing wound healing strategies.

This review explores the application of induced pluripotent stem cells (iPSCs) in regenerative medicine. The therapeutic significance of iPSC-derived cell therapy within regenerative medicine, emphasizes their reprogramming process and crucial role in cellular differentiation while setting the purpose and scope for the comprehensive exploration of iPSC-derived cell therapy. The subsequent sections intricately examine iPSC-derived cell therapy, unraveling the diverse derivatives of iPSCs and striking a delicate balance between advantages and limitations in therapeutic applications. Mechanisms of action, revealing how iPSC-derived cells seamlessly integrate into tissues, induce regeneration, and contribute to disease modeling and drug screening advancements is discussed. The analysis extends to clinical trials, shedding light on outcomes, safety considerations, and ethical dimensions. Challenges and concerns, including the risk of tumorigenesis and scalability issues, are explored. The focus extends to disease-specific applications, showcasing iPSC-derived cell therapy as a promising avenue for various medical conditions, supported by illustrative case studies. Future directions and research needs are outlined, identifying areas for further exploration, safety considerations and potential enhancements that will shape the future landscape of iPSC-derived therapies. In conclusion, this review provides a significant understanding of iPSC-derived cell therapy's status that contemplates the implications for regenerative medicine and personalized treatment using iPSCs, offering a comprehensive perspective on the evolving field within the confines of a dynamic and promising scientific frontier.

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).

Rheumatoid arthritis (RA) affects millions of people worldwide, but there are limited drugs available to treat it, so acquiring a more comprehensive comprehension of the underlying reasons and mechanisms behind inflammation is crucial, as well as developing novel therapeutic approaches to manage it and mitigate or forestall associated harm. It is evident that current in vitro models cannot faithfully replicate all aspects of joint diseases, which makes them ineffective as tools for disease research and drug testing. Organ-on-a-chip (OoC) technology is an innovative platform that can mimic the microenvironment and physiological state of living tissues more realistically than traditional methods by simulating the spatial arrangement of cells and interorgan communication. This technology allows for the precise control of fluid flow, nutrient exchange, and the transmission of physicochemical signals, such as bioelectrical, mechanical stimulation and shear force. In addition, the integration of cutting-edge technologies like sensors, 3D printing, and artificial intelligence enhances the capabilities of these models. Here, we delve into OoC models with a particular focus on Synovial Joints-on-a-Chip, where we outline their structure and function, highlighting the potential of the model to advance our understanding of RA. We integrate the actual evidence regarding various OoC models and their possible integration for multisystem disease study in RA research for the first time and introduce the prospects and opportunities of the chip in RA etiology and pathological mechanism research, drug research, disease prevention and human precision medicine. Although many challenges remain, OoC holds great promise as an in vitro model that approaches physiology and dynamics.

Gene therapy utilizes nucleic acid drugs to treat diseases, encompassing gene supplementation, gene replacement, gene silencing, and gene editing. It represents a distinct therapeutic approach from traditional medications and introduces novel strategies for genetic disorders. Over the past two decades, significant advancements have been made in the field of gene therapy, leading to the approval of various gene therapy drugs. Gene therapy was initially employed for treating genetic diseases and cancers, particularly monogenic conditions classified as orphan diseases due to their low prevalence rates; however, polygenic or complex diseases exhibit higher incidence rates within populations. Extensive research on the etiology of polygenic diseases has unveiled new therapeutic targets that offer fresh opportunities for their treatment. Building upon the progress achieved in gene therapy for monogenic diseases and cancers, extending its application to polygenic or complex diseases would enable targeting a broader range of patient populations. This review aims to discuss the strategies of gene therapy, methods of gene editing (mainly CRISPR-CAS9), and carriers utilized in gene therapy, and highlight the applications of gene therapy in polygenic or complex diseases focused on applications that have either entered clinical stages or are currently undergoing clinical trials.

Tumor microenvironment (TME) heterogeneity leads to a discrepancy in survival prognosis and clinical treatment response for patients with HCC. The clinical applications of documented molecular subtypes are constrained by several issues.

We integrated 3 single-cell data sets to describe the TME landscape and identified 6 prognosis-related cell subclusters. Unsupervised clustering of subcluster-specific markers was performed to generate transcriptomic subtypes. The predictive value of these molecular subtypes for prognosis and treatment response was explored in multiple external HCC cohorts and the Xiangya HCC cohort. TME features were estimated using single-cell immune repertoire sequencing, mass cytometry, and multiplex immunofluorescence. The prognosis-related score was constructed based on a machine-learning algorithm. Comprehensive single-cell analysis described TME heterogeneity in HCC. The 5 transcriptomic subtypes possessed different clinical prognoses, stemness characteristics, immune landscapes, and therapeutic responses. Class 1 exhibited an inflamed phenotype with better clinical outcomes, while classes 2 and 4 were characterized by a lack of T-cell infiltration. Classes 5 and 3 indicated an inhibitory tumor immune microenvironment. Analysis of multiple therapeutic cohorts suggested that classes 5 and 3 were sensitive to immune checkpoint blockade and targeted therapy, whereas classes 1 and 2 were more responsive to transcatheter arterial chemoembolization treatment. Class 4 displayed resistance to all conventional HCC therapies. Four potential therapeutic agents and 4 targets were further identified for high prognosis-related score patients with HCC.

Our study generated a clinically valid molecular classification to guide precision medicine in patients with HCC.

CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQR and enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.

The fact that certain oral carcinoma patients experience radiotherapy failure implies that a more radioresistant and aggressive phenotype of surviving cancer cells potentially occurs during treatment. Our study aimed to establish radioresistant oral cancer cells through a fractionated irradiation protocol that mimics clinically relevant radiotherapy dosing strategies and to investigate all-round alterations in the malignant phenotype.

Radioresistant oral carcinoma cells were generated by exposing Cal27 and Detroit 562 cells to 60 Gy radiation in 10 dose-escalating fractions and verified by cell immunofluorescence. Specific markers related to the epithelial-mesenchymal transition (EMT) process and the cancer stem cell (CSC) phenotype were assessed by Western blotting. Cell invasion and migration were evaluated using Matrigel-coated transwell and wound healing assays, respectively. Nontargeted metabolomics was used to mechanistically delineate the potential metabolic patterns linked to EMT and CSCs; the CSC phenotype was also examined by sphere formation assays and cell immunofluorescence.

Radioresistant oral carcinoma cell lines were successfully established and validated. These cells exhibited enhanced EMT and increase in both cell invasion and migration. These radioresistant cells further demonstrated a high metabolic profile, notably marked by lipid metabolism reprogramming and functional enrichment of ATP-binding cassette (ABC) transporters. Consistently, enhanced CSC phenotype in radioresistant cells was confirmed by elevated expression of stemness markers and increased sphere-forming capacity.

Radioresistant oral carcinoma cells subjected to fractionated radiation exhibit an augmented malignant phenotype. The metabolic characteristics linked to enhanced EMT and CSC phenotypes provide potential targets for improving radiotherapy in oral carcinoma.

Mutations in the C-terminal of KIF1A (Kinesin family member 1A) may lead to amyotrophic lateral sclerosis (ALS) through unknown mechanisms that are not yet understood. Using iPSC reprogramming technology and motor neuron differentiation techniques, we generated iPSCs from a healthy donor and two ALS patients with KIF1A mutations (R1457Q and P1688L) and differentiated them into spinal motor neurons (iPSC-MN) to investigate KIF1A-related ALS pathology. Our in vitro iPSC-iMN model faithfully recapitulated specific aspects of the disease, such as neurite fragmentation. Through this model, we observed that these mutations led to KIF1A aggregation at the proximal axon of motor neurons and abnormal accumulation of its transport cargo, LAMP1, resulting in autophagy dysfunction and cell death. RNAseq analysis also indicated that the functions of the extracellular matrix, structure, and cell adhesion were significantly disturbed. Notably, using rapamycin during motor neuron differentiation can effectively prevent motor neuron death.

The immune system, functioning as the body's "defense army", plays a role in surveillance, defense. Any disruptions in immune system can lead to the development of immune-related diseases. Extensive researches have demonstrated the crucial immunoregulatory role of mesenchymal stem cells (MSCs) in these diseases. Of particular interest is the ability to induce somatic cells under specific conditions, generating a new cell type with stem cell characteristics known as induced pluripotent stem cell (iPSC). The differentiation of iPSCs into MSCs, specifically induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), hold promise as a potential solution to the challenges of MSCs, potentially serving as an alternative to traditional drug therapies. Moreover, the products of iMSCs, termed induced pluripotent stem cell-derived mesenchymal stem cell-derived extracellular vesicles (iMSC-EVs), may exhibit functions similar to iMSCs. With the biological advantages of EVs, they have become the focus of "cell-free therapy". Here, we provided a comprehensive summary of the biological impact of iMSCs on immune cells, explored the applications of iMSCs and iMSC-EVs in diseases, and briefly discussed the fundamental characteristics of EVs. Finally, we overviewed the current advantages and challenges associated with iMSCs and iMSC-EVs. It is our hope that this review related to iMSCs and iMSC-EVs will contribute to the development of new approaches for the treatment of diseases.

Mutations in the DMD gene lead to Duchenne muscular dystrophy (DMD), a severe neuromuscular disorder affecting young boys as they acquire motor functions. DMD is typically diagnosed at 2-4 years of age, but the absence of dystrophin has negative impacts on skeletal muscles before overt symptoms appear in patients, which poses a serious challenge in current standards of care. Here, we investigated the consequences of dystrophin deficiency during skeletal muscle development. We used single-cell transcriptome profiling to characterize the myogenic trajectory of human pluripotent stem cells and showed that DMD cells bifurcate to an alternative branch when they reach the somite stage. Dystrophin deficiency was linked to marked dysregulations of cell junction proteins involved in the cell state transitions characteristic of embryonic somitogenesis. Altogether, this work demonstrates that in vitro, dystrophin deficiency has deleterious effects on cell-cell communication during myogenic development, which should be considered in future therapeutic strategies for DMD.

Diabetic wounds are characterized by incomplete healing and delayed healing, resulting in a considerable global health care burden. Exosomes are lipid bilayer structures secreted by nearly all cells and express characteristic conserved proteins and parent cell-associated proteins. Exosomes harbor a diverse range of biologically active macromolecules and small molecules that can act as messengers between different cells, triggering functional changes in recipient cells and thus endowing the ability to cure various diseases, including diabetic wounds. Exosomes accelerate diabetic wound healing by regulating cellular function, inhibiting oxidative stress damage, suppressing the inflammatory response, promoting vascular regeneration, accelerating epithelial regeneration, facilitating collagen remodeling, and reducing scarring. Exosomes from different tissues or cells potentially possess functions of varying levels and can promote wound healing. For example, mesenchymal stem cell-derived exosomes (MSC-exos) have favorable potential in the field of healing due to their superior stability, permeability, biocompatibility, and immunomodulatory properties. Exosomes, which are derived from skin cellular components, can modulate inflammation and promote the regeneration of key skin cells, which in turn promotes skin healing. Therefore, this review mainly emphasizes the roles and mechanisms of exosomes from different sources, represented by MSCs and skin sources, in improving diabetic wound healing. A deeper understanding of therapeutic exosomes will yield promising candidates and perspectives for diabetic wound healing management.

With the use of specific genetic factors and recent developments in cellular reprogramming, it is now possible to generate lineage-committed cells or induced pluripotent stem cells (iPSCs) from readily available and common somatic cell types. However, there are still significant doubts regarding the safety and effectiveness of the current genetic methods for reprogramming cells, as well as the conventional culture methods for maintaining stem cells. Small molecules that target specific epigenetic processes, signaling pathways, and other cellular processes can be used as a complementary approach to manipulate cell fate to achieve a desired objective. It has been discovered that a growing number of small molecules can support lineage differentiation, maintain stem cell self-renewal potential, and facilitate reprogramming by either increasing the efficiency of reprogramming or acting as a genetic reprogramming factor substitute. However, ongoing challenges include improving reprogramming efficiency, ensuring the safety of small molecules, and addressing issues with incomplete epigenetic resetting. Small molecule iPSCs have significant clinical applications in regenerative medicine and personalized therapies. This review emphasizes the versatility and potential safety benefits of small molecules in overcoming challenges associated with the iPSCs reprogramming process.

Lactate, a metabolic byproduct, has gained recognition as a highly influential signaling molecule. Lactylation, an emerging form of post-translational modification derived from lactate, plays a crucial role in numerous cellular processes such as inflammation, embryonic development, tumor proliferation, and metabolism. However, the precise molecular mechanisms through which lactylation governs these biological functions in both physiological and pathological contexts remain elusive. Hence, it is imperative to provide a comprehensive overview of lactylation in order to elucidate its significance in biological processes and establish a foundation for forthcoming investigations. This review aims to succinctly outline the process of lactylation modification and the characterization of protein lactylation across diverse organisms. Additionally, A summary of the regulatory mechanisms of lactylation in cellular processes and specific diseases is presented. Finally, this review concludes by delineating existing research gaps in lactylation and proposing primary directions for future investigations.

Cartilage, an important connective tissue, provides structural support to other body tissues, and serves as a cushion against impacts throughout the body. Found at the end of the bones, cartilage decreases friction and averts bone-on-bone contact during joint movement. Therefore, defects of cartilage can result from natural wear and tear, or from traumatic events, such as injuries or sudden changes in direction during sports activities. Overtime, these cartilage defects which do not always produce immediate symptoms, could lead to severe clinical pathologies. The emergence of induced pluripotent stem cells (iPSCs) has revolutionized the field of regenerative medicine, providing a promising platform for generating various cell types for therapeutic applications. Thus, chondrocytes differentiated from iPSCs become a promising avenue for non-invasive clinical interventions for cartilage injuries and diseases. In this review, we aim to highlight the current strategies used for in vitro chondrogenic differentiation of iPSCs and to explore their multifaceted applications in disease modeling, drug screening, and personalized regenerative medicine. Achieving abundant functional iPSC-derived chondrocytes requires optimization of culture conditions, incorporating specific growth factors, and precise temporal control. Continual improvements in differentiation methods and integration of emerging genome editing, organoids, and 3D bioprinting technologies will enhance the translational applications of iPSC-derived chondrocytes. Finally, to unlock the benefits for patients suffering from cartilage diseases through iPSCs-derived technologies in chondrogenesis, automatic cell therapy manufacturing systems will not only reduce human intervention and ensure sterile processes within isolator-like platforms to minimize contamination risks, but also provide customized production processes with enhanced scalability and efficiency.

Ephrin receptor A2 (EphA2), a member of the Ephrin receptor family, is closely related to the progression of oral squamous cell carcinoma (OSCC). Cancer stem cells (CSCs) play essential roles in OSCC development and occurrence. The underlying mechanisms between EphA2 and CSCs, however, are not yet fully understood. Here, we found that EphA2 was overexpressed in OSCC tissues and was associated with poor prognosis. Knockdown of EphA2 dampened the CSC phenotype and the tumour-initiating frequency of OSCC cells. Crucially, the effects of EphA2 on the CSC phenotype relied on KLF4, a key transcription factor for CSCs. Mechanistically, EphA2 activated the ERK signalling pathway, promoting the nuclear translocation of YAP. Subsequently, YAP was bound to TEAD3, leading to the transcription of KLF4. Overall, our findings revealed that EphA2 can enhance the stemness of OSCC cells, and this study identified the EphA2/KLF4 axis as a potential target for treating OSCC.

There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac).

We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks.

The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone.

The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications.

Patient-specific, human-based cellular models integrating a biomimetic blood-brain barrier (BBB), immune, and myelinated neuron components are critically needed to enable accelerated, translationally relevant discovery of neurological disease mechanisms and interventions. By engineering a novel brain-mimicking 3D hydrogel and co-culturing all six major brain cell types derived from patient iPSCs, we have constructed, characterized, and utilized a multicellular integrated brain (miBrain) immuno-glial-neurovascular model with in vivo- like hallmarks inclusive of neuronal activity, functional connectivity, barrier function, myelin-producing oligodendrocyte engagement with neurons, multicellular interactions, and transcriptomic profiles. We implemented the model to study Alzheimer's Disease pathologies associated with APOE4 genetic risk. APOE4 miBrains differentially exhibit amyloid aggregation, tau phosphorylation, and astrocytic GFAP. Unlike the co-emergent fate specification of glia and neurons in organoids, miBrains integrate independently differentiated cell types, a feature we harnessed to identify that APOE4 in astrocytes promotes neuronal tau pathogenesis and dysregulation through crosstalk with microglia.

Glycogen Storage disease type 4 (GSD4), a rare disease caused by glycogen branching enzyme 1 (GBE1) deficiency, affects multiple organ systems including the muscles, liver, heart, and central nervous system. Here we report a GSD4 patient, who presented with severe hepatosplenomegaly and cardiac ventricular hypertrophy. GBE1 sequencing identified two variants: a known pathogenic missense variant, c.1544G>A (p.Arg515His), and a missense variant of unknown significance (VUS), c.2081T>A (p. Ile694Asn). As a liver transplant alone can exacerbate heart dysfunction in GSD4 patients, a precise diagnosis is essential for liver transplant indication. To characterize the disease-causing variant, we modeled patient-specific GBE1 deficiency using CRISPR/Cas9 genome-edited induced pluripotent stem cells (iPSCs).

iPSCs from a healthy donor (iPSC-WT) were genome-edited by CRISPR/Cas9 to induce homozygous p.Ile694Asn in GBE1 (iPSC-GBE1-I694N) and differentiated into hepatocytes (iHep) or cardiomyocytes (iCM). GBE1 enzyme activity was measured, and PAS-D staining was performed to analyze polyglucosan deposition in these cells.

iPSCGBE1-I694N differentiated into iHep and iCM exhibited reduced GBE1 protein level and enzyme activity in both cell types compared to iPSCwt. Both iHepGBE1-I694N and iCMGBE1-I694N showed polyglucosan deposits correlating to the histologic patterns of the patient's biopsies.

iPSC-based disease modeling supported a loss of function effect of p.Ile694Asn in GBE1. The modeling of GBE1 enzyme deficiency in iHep and iCM cell lines had multi-organ findings, demonstrating iPSC-based modeling usefulness in elucidating the effects of novel VUS in ultra-rare diseases.