Neuroinflammation plays a fundamental role in several neurodegenerative diseases, including Alzheimer's disease (AD), the leading cause of dementia worldwide. As the main defense response of the central nervous system (CNS), neuroinflammation can be either protective or detrimental depending on the stage of the disease. The pivotal role of neuroinflammation in AD has led to increasing investigations into neuroinflammatory mechanisms, aiming to develop AD-modifying therapies. A significant advance in the field was the emergence of the human induced pluripotent stem cell (hiPSC) model, enabling the study of patient-derived cells. Moreover, the development of hiPSC-derived brain organoids, which mimic specific aspects of the human CNS, has expanded our understanding of neuroinflammation in AD. Here, we review how AD organoid models have evolved, focusing on the integration of microglia-the brain's primary immune surveillance cells. We also summarize recent findings on how glial activation and the crosstalk between microglia and other CNS cells affect AD progression. Lastly, we address the potential of hiPSC-derived organoids as a preclinical model for screening AD drugs.

Rotator cuff repair often results in scar tissue rather than tendon regeneration, leading to inferior strength and high re-tear risk. This study evaluated the regenerative efficacy of an injectable formulation combining adipose-derived stem cell (ADSC) clusters with collagen in an animal model.

Gastrointestinal diseases often involve cellular damage, degeneration or dysfunction in the tract, frequently requiring surgical interventions risking complications and lowered quality of life. Regenerative medicine holds great promise in improving patient care and providing novel treatment options for previously irreparable and untreatable tissues. Despite the clinical potential of intestinal organoids as a resource for regenerative cell therapy and bioengineering, the lack of clinical-grade cultures has hampered further development. Moreover, strategies to efficiently and reliably expand clinical-grade cultures at the scale required for application is limited.

Type 2 diabetes (T2D) and its complications represent a complex disorder involving multiple pathophysiological processes. Although conventional therapeutic approaches partially regulate blood glucose, they fail to fundamentally reverse disease progression or effectively prevent complications. This review summarizes the current research advance and challenges of using different forms of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in treating T2D and complications. It begins with an introduction to the characteristics of MSC-EVs. Subsequently, the mechanisms and therapeutic prospects of natural MSC-EVs are analyzed, with a focus on their roles in inflammatory modulation, tissue regeneration, and improving insulin resistance. Engineering MSC-EVs, covering strategies including optimizing MSC culture conditions, modifying EV contents, and establishing MSC-EV delivery systems based on bioactive materials are then discussed, which boost EV yield and quality while enhancing therapeutic efficacy. Current challenges, including the limited yield and high heterogeneity of natural MSC-EVs, as well as issues related to long-term safety, immunocompatibility, and large-scale production of engineered MSC-EVs are finally overviewed, with emphasizing artificial intelligence in guiding future research directions. These summaries are crucial for clinical translation of MSC-EVs and will ultimately provide T2D patients with an effective and safe treatment option.

Osteoporosis (OP) is a metabolic bone disease characterized by low bone mineral density (BMD), and its pathogenesis involves endoplasmic reticulum (ER) stress-related cell death. This study aimed to identify diagnostic biomarkers associated with ER stress-related cell death in OP and explore their underlying mechanisms. The training dataset (GSE56815), validation dataset (GSE56814), and single-cell RNA sequencing (scRNA-seq) dataset (GSE147287) were downloaded. Differentially expressed genes (DEGs) between OP patients and controls were identified. Candidate genes were obtained by intersecting DEGs with ER stress-related genes and programmed cell death (PCD)-related genes. Machine learning was used to screen intersection genes, and biomarkers were determined via expression level analysis. Gene set enrichment analysis (GSEA), immune cell infiltration analysis, drug prediction and molecular docking, scRNA-seq analysis, key cell screening, cell communication analysis, and pseudotime analysis were performed. Finally, reverse transcription quantitative polymerase chain reaction (RT-qPCR) were further conducted. A total of 28 candidate genes were obtained by intersection. CAMKK2 and DAPK3 were confirmed as biomarkers, and were consistently down-regulated in both datasets and verified by RT-qPCR. GSEA analysis revealed that biomarkers were enriched in cytokine-cytokine receptor interaction. Correlations between biomarkers and activated dendritic cells were found via immune cell infiltration analysis. Preliminary computational analyses indicated that drugs including calcitriol and danazol may potentially interact with the biomarkers in a stable manner. Bone marrow-derived mesenchymal stem cells (BM-MSCs) were identified as potential key cells via scRNA-seq analysis. Complex interactions involving BM-MSCs, such as ANGPTL4-CDH11 mediating BM-MSC self-communication, were revealed by cell communication analysis. Dynamic expression of biomarkers during BM-MSC differentiation was shown by pseudotime analysis: CAMKK2 fluctuated with differentiation stages, while DAPK3 shifted from high to low then high expression. CAMKK2 and DAPK3 were confirmed as diagnostic biomarkers for OP, providing insights into OP diagnosis and potential therapeutic targets.

Tissue engineering offers great promise for regenerating damaged organs including the heart. Although direct attachment of grafts at the target site is possible during surgery, minimally invasive delivery and suture-free approaches could reduce patient discomfort and allow repeat administration. However, for the therapy to be effective it is essential that the graft is successfully delivered to the epicardium and retained on target. Here, methacrylated alginate-based shape-memory patches labelled with 111InCl3 and loaded with luciferase expressing stem-cells were either injected towards the epicardium under ultrasound guidance or surgically grafted onto mouse hearts. Patch and cell location were serially tracked using SPECT-CT and bioluminescence imaging. Radiolabelling of shape-memory patches permitted serial tracking of graft location for seven days in-vivo, and revealed that injected patches rarely attached on-target whilst surgically implanted patches rapidly detached from the epicardium. In-vivo imaging was then used to evaluate modifications to biomaterial formulation and patch attachment strategies. This ultimately resulted in effective, suture-free surgical attachment of chitosan-coated patches loaded with luciferase-expressing human embryonic stem cell-derived epicardial cells onto the heart, illustrating a model therapeutic. This translational imaging approach facilitates iterative optimization of a novel biomaterial and could have wide-reaching applications for enhancing a range of regenerative therapies.

Embryonic stem cells (ESCs) constitute a unique lab resource, as they can differentiate to all other cell types, making them pluripotent. We established cynomolgus ESCs using ICSI-derived blastocysts. Cynomolgus ESCs expressed pluripotent markers in immunostaining and fluorescence-activated cell sorting analyses. To investigate its differentiation potential, we confirmed the expression of layer markers from the three germ layers after embryonic body differentiation. Moreover, our ESC shows a normal genotype and no mycoplasma contamination.

This protocol generates neural assembloids from human induced pluripotent stem cells (hiPSCs) to model human brain circuitry. It details the differentiation of excitatory-predominant hippocampal (Hc) and cortical (Cx) organoids and their fusion with inhibitory interneuron-predominant ganglionic eminence (GE) organoids. We then detail procedures for maintaining assembloids to promote interneuron migration and network integration, followed by functional assessment using two-photon calcium imaging to measure neuronal activity. For complete details on the use and execution of this protocol, please refer to McCrimmon et al.1.

Metanephric mesenchymal cells (MMCs) hold therapeutic potential for acute kidney injury (AKI), but their efficacy is limited, and the mechanisms underlying their action remain unclear. This study aimed to investigate whether catalpol-pretreated MMCs (MMCs-cata) could enhance the efficacy of AKI treatment by regulating key signaling pathways.

Psoriasis is a refractory immune-related disease. In recent years, it has been discovered that mesenchymal stem cells (MSCs) can be used as a new therapeutic approach for psoriasis, but their potential therapeutic mechanism remains unclear. This study aims to explore the role of MSCs in the treatment of psoriasis.

Invariant natural killer T (iNKT) cells, upon activation, exhibit antitumor roles by bridging innate and acquired immunity. To overcome the challenges in producing iNKT cells from patients with cancer, we previously developed allogeneic human induced pluripotent stem cell-derived iNKT (iPSC-iNKT) cells. However, the activation of iPSC-iNKT cells by glycolipid ligands remains a critical step for iNKT cell-mediated cancer therapy. To show the effect of iPSC-iNKT cell-mediated antitumor immunity, in this preclinical study, by taking advantage of a human immune cell-transplanted patient-derived xenograft model using human IL-7/15 knock-in NSG mice, we demonstrate that a combination of iPSC-iNKT cells and α-galactosylceramide-pulsed antigen-presenting cells (αGalCer/APC) induces robust antitumor effects. Single-cell analysis of tumor-infiltrating lymphocytes revealed that this combination therapy uniquely expanded tumor-reactive memory-phenotype CD4 and CD8 T cells. Taken together, upon activation by αGalCer/APC, iPSC-iNKT cells are capable of effectively inducing antitumor T cell immunity, making them a promising tool for generating personalized antitumor T cell immunity.

Cornelia de Lange syndrome (CdLS) is a rare genetic disorder that affects almost any organ, including the central nervous system. It leads to a wide range of neurodevelopmental delays, and there are currently no available clinical treatments. CdLS is caused by pathogenic variants in one of the 7 genes coding for the cohesin complex, a multimeric structure responsible for sister chromatid cohesion, or for cohesin ring-interacting proteins. Additionally, altered regulation of molecular pathways during development, including the canonical WNT pathway, can cause CdLS malformations. In our study, we evaluated the positive effects of using lithium as an activator of the canonical WNT pathway to ameliorate neural CdLS phenotype. We have exploited accurate two-dimensional (2D) and three-dimensional (3D) human central nervous system in vitro models representing disease-related neurobiological phenotypes: induced pluripotent stem cells of human origin (hiPSCs) differentiated into neural precursors, neurons, and brain organoids (BOs). CdLS models demonstrate alterations in proliferation and differentiation capabilities when mimicking HDAC8 haploinsufficiency. Furthermore, RNA-seq analysis of BOs revealed that both neuronal differentiation and the WNT pathway are downregulated when treated with the HDAC8 inhibitor alone. Following lithium treatment, cells show an enhanced ability to differentiate into the neuronal lineage. Additionally, our working hypothesis is that a specific mechanism may exist that, by connecting lipid metabolism, canonical WNT pathway, and cell death, results in typical CdLS neurodevelopmental deficits.

Lung cancer, being the top cause of global cancer-related mortality, calls for effective treatments. RN7SK (7SK) is a long non-coding RNA (lncRNA) that plays a significant role in the regulation of gene transcription and thereby controls essential cellular activities. Limited evidence supports the anticancer potential of 7SK, and its suppressive effects have not been tested against lung cancer. This study explored the anticancer effects of RN7SK (7SK), a long non-coding RNA known to regulate gene transcription, through exosome-mediated delivery in lung cancer cells. Treatment with exosome-loaded 7SK (Exo-7SK) significantly elevated 7SK levels in non-small-cell lung cancer cells and suppressed key cancer traits. Exo-7SK reduced cell viability and proliferation, promoted apoptosis, and inhibited migration and invasion by shifting gene expression away from epithelial-mesenchymal transition. It also impaired spheroid formation and reduced spheroid dispersion and viability in 3D microfluidic cultures. In conclusion, our findings highlight the cancer-suppressive potential of exosome-mediated 7SK delivery against lung cancer, demonstrating significant efficacy in both 2D and 3D culture models. These observations warrant further confirmation in future studies employing advanced designs and clinically relevant models.

Paneth cell metaplasia (PCM) is a phenomenon in which Paneth cells, typically found in the small intestine, appear in the colonic epithelium of patients with ulcerative colitis (UC). Our study demonstrates that the PCM occurrence correlates with disease duration and active inflammation. Furthermore, we identified IL-22, an inflammation-associated cytokine, as a key regulator that promotes PCM formation in the colonic epithelium through suppression of Notch signaling and induces REG3A expression within metaplastic niches. In vitro, we show that Reg3a directly enhances cell proliferation and promotes wound healing using mouse colonic organoids. In vivo, Reg3aΔIEC mice in both acute and chronic DSS-induced colitis models exhibit delayed wound healing. Additionally, studies conducted with patient-derived human colonic organoids revealed that REG3A administration stimulates cell proliferation and accelerates wound healing. Together, these findings support a protective role of PCM-associated REG3A in the colonic epithelium of patients with UC.

Disabling hearing impairment is a common human sensory deficit. OTOF is a major deafness gene. It codes for the synaptic protein otoferlin and is essential for transmitter release by inner hair cells (IHCs). Upon genetic loss of otoferlin, cochlear structure and function remain intact up to the IHC synapses, which fail to encode sound. Building on preclinical hearing restoration by AAV-mediated cochlear gene transfer in mice, clinical OTOF-gene-therapy trials are now targeting the pediatric population. However, preclinical optimization and characterization remain urgent needs for the development of OTOF-gene-therapy. Here, we report on the generation and characterization of a marmoset KO that models OTOF-related auditory synaptopathy and can thus address these needs. Following ovary stimulation, harvesting, in vitro maturation and fertilization of oocytes, we injected the zygotes with Cas9 and guide RNAs to disrupt OTOF. Mutant embryos were transferred into the uterus of foster mothers. Marmosets with biallelic, non-mosaic OTOF-KO were normally born and raised by their respective foster parents. Auditory brainstem recordings and otoacoustic emissions revealed profound auditory synaptopathy and OTOF-KO was further validated by the lack of otoferlin expression in IHCs. The new non-human primate model of OTOF-related auditory synaptopathy will serve studies of specificity, efficacy, and longevity of novel inner ear therapies.

Genetically modified mesenchymal stem cells (MSCs) have been shown to enhance their therapeutic properties, offering more effective treatment options for various diseases, including metabolic associated fatty liver disease (MASLD). The m7G methyltransferase METTL1 plays a critical role in regulating RNA splicing, stability, and translation. This study presents our findings on METTL1 modified human umbilical cord MSCs, emphasizing their therapeutic effects and the mechanisms involved in treating MASLD.

Camizestrant, a next-generation selective estrogen receptor (ER) degrader and complete ER antagonist, has been associated with a reversible dose- and time-dependent heart rate (HR) reduction in clinical studies. This nonclinical investigation aimed to understand the mechanism of camizestrant-induced HR reduction. The effects of camizestrant on HR in vivo were assessed in rat and dog telemetry studies. Effects on pacemaker channel function in vitro were assessed using patch-clamp electrophysiology in Chinese hamster ovary cells expressing human hyperpolarization-activated cyclic nucleotide-gated channel 4 (hHCN4), human embryonic stem cell (hESC)-derived sinoatrial node (SAN) cardiomyocytes, and primary rat SAN cardiomyocytes. In dogs, 28-day repeat-dose camizestrant administration caused a reversible dose- and time-dependent HR reduction (maximum reduction of 53 beats per min [bpm] on Day 25 vs pre-study levels at 20 mg/kg). HR reduction was also noted in rats (maximum reduction 89 bpm vs vehicle [23%] on Day 5 of a 7-day study at 75 mg/kg). Responses to chronotropic stimuli (e.g., atropine and isoprenaline) were reduced in dogs treated with camizestrant. Camizestrant-induced HR reduction was still present following combined sympathetic (atenolol) and parasympathetic (atropine) inhibition in dogs, as well as vagotomy in rats. Camizestrant reduced hHCN4 current density in Chinese hamster ovary cells, as well as beat rate and "funny" pacemaker (If) current activity in hESC-derived SAN cardiomyocytes. Camizestrant at 75 mg/kg for 7 days significantly reduced If current activity versus vehicle in isolated SAN cardiomyocytes. These results support the hypothesis that camizestrant exerts a pharmacologic, reversible reduction in HR by decreasing SAN pacemaker current activity.