Human intestinal Organoids (HIOs) may be set up from primary human tissue following dissociation, release, and culture of intestinal LGR5 + stem cells. Here, we characterise the use of a semi-automated mechanical tissue dissociation technique, which demonstrates an expedited workflow and streamlined protocol, standardising key aspects of the process while consistently maintaining a high level of cultured organoid viability.

The capacity to regenerate skeletal muscle after injury requires a complex and well-coordinated cellular response, which is challenged in aged skeletal muscle. Here, we unravel the intricate dynamics of elderly human skeletal muscle regeneration by combining spatial, temporal, and single cell transcriptomics. Using spatial RNA sequencing (n = 3), we profile the expression of human protein-coding genes in elderly human skeletal muscle biopsies before as well as 2-, 8-, and 30-day post injury (NCT03754842). Single Cell-Spatial deconvolution analysis highlights monocytes/macrophages and fibro-adipogenic progenitors (FAPs) as pivotal players in human muscle regeneration. By utilizing flow cytometry (n = 9) and cell sorting we confirm the increased cellular content and activity during regeneration. Spatial correlation analysis unveils FAPs and monocytes/macrophages co-localization and intercellular communication, mediated by complement factor C3. Immunostaining confirms C3 expression in FAPs and FAP secretion of C3, suggesting a role in phagocytosis of necrotic muscle cells. Finally, functional assays demonstrate C3's impact on human monocyte metabolism, survival and phagocytosis, unveiling its involvement in skeletal muscle regeneration. These insights elucidate the FAP-macrophage interplay in aged human muscle with perspectives for future therapeutic interventions to reduce the age-induced decline in regenerative capacity.

Salivary gland fibrosis results in salivary hypofunction whose current treatments are palliative. Mesenchymal stem/stromal cells (MSCs) are anti-fibrotic and anti-inflammatory and provide a promising alternative to treat fibrosis, but face translational challenges due to poor tissue targeting and retention. Scaffolds could facilitate targeted MSC delivery and improve MSC retention. Here, we demonstrated the feasibility of using stromal extracellular matrix (ECM)-mimicking elastin-alginate cryoelectrospun scaffolds (CES) for MSC delivery. Using MSC-like primary embryonic salivary mesenchyme cells (SMSCs), we demonstrated that CES supported viable, healthy and non-fibrotic long-term stromal cell maintenance while suppressing the fibrotic phenotype in myofibroblasts. We established an in vitro fibrosis model by coculturing SMSCs and myofibroblasts on CES and validated that SMSCs on CES imparted anti-fibrotic effects on myofibroblasts; however, these effects were suppressed by TGF-β1. In vivo orthotopic implantation of CES and SMSC-CES constructs showed that CES were biocompatible, non-inflammatory, and non-fibrotic, however, they did not remediate fibrosis. Further, supplementation of anti-fibrotic FGF2 to the in vitro fibrosis model demonstrated partial rescue of the anti-fibrotic effects of SMSC-CES constructs on myofibroblasts stimulated by TGF-β1 signaling. These studies set the premise for multi-pronged therapeutic approaches and validate CES as a potential stromal cell delivery vehicle in regenerative medicine applications.

β-propeller protein-associated neurodegeneration (BPAN) is a rare X-linked neurodegenerative disorder caused by mutations in the WDR45 gene, yet its molecular mechanisms remain poorly understood. Here, we identify a role for WDR45 in stress granule (SG) disassembly, mediated through its phase separation with Caprin-1. We demonstrate that WDR45 forms gel-like condensates via its WD5 domain, which competitively displaces G3BP1 from Caprin-1 to promote SG disassembly. BPAN-associated WDR45 mutations impair condensate formation and Caprin-1 interaction, leading to delayed SG disassembly, which correlates with earlier disease onset. WDR45 depletion also exacerbates amyotrophic lateral sclerosis-associated pathological SGs, highlighting its broader relevance to neurodegenerative diseases. Using iPSC-derived midbrain neurons from a BPAN patient, we demonstrate delayed SG recovery, directly linking WDR45 dysfunction to neurodegeneration. These findings establish WDR45 as a critical regulator of SG dynamics, uncover a potential molecular basis of BPAN pathogenesis, and identify therapeutic targets for neurodegenerative diseases associated with SG dysregulation.

Vero cells, as approved by the World Health Organization, have been the most commonly used continuous cell line for viral vaccine production over the last 25 years, but their adherent phenotype continues to limit productivity. Adapting to a suspension culture would overcome this restriction and reduce production costs. First, a Vero suspension isolate was obtained and metabolically characterized. Second, RNA sequencing analysis was used to identify differentially expressed genes between adherent and suspension cells, which revealed complete downregulation of adhesion and matrix-associated genes. Additionally, signaling pathways involving Wnt and other tyrosine kinase receptors were identified as potential leads for growth optimization. In particular, supplementation with fibroblast growth factor 2 allowed for a 20% increase in cell density. Finally, a comparative viral productivity assay revealed a 30% increase in poliovirus production in suspension Vero cells compared to adherent cells depending on the serotype, as well as a 140% increase in respiratory syncytial virus production and a 150% increase in yellow fever virus production. This work establishes the potential of the suspension Vero cell line as a new cell platform for viral vaccine production.

Relapsed and/or refractory acute myeloid leukemia (AML) post-allogeneic hematopoietic cell transplantation (HCT) is usually fatal. We previously reported that post-HCT immunotherapy with Epstein-Barr virus (EBV)-specific donor CD8+ T cells engineered to express a Wilms Tumor Antigen 1-specific T-cell receptor (TTCR-C4) appeared to prevent relapse in high-risk patients. In this phase I/II clinical trial (NCT01640301), we evaluated safety (primary endpoint), persistence and efficacy (secondary endpoints) of EBV- or Cytomegalovirus (CMV)-specific TTCR-C4 in fifteen patients with active AML post-HCT. Infusions were well tolerated, with no dose-limiting toxicities or serious adverse events related to the product. However, TTCR-C4 cells did not clearly improve outcomes despite EBV-specific TTCR-C4 cells showing enhanced potential for prolonged persistence compared to CMV-specific TTCR-C4. Investigating the fate of persisting TTCR-C4, we identified a shift towards natural killer-like (NKL) terminal differentiation, distinct from solid tumor-associated canonical exhaustion programs. In one patient, treatment with azacitidine appeared to mitigate this NKL skewing, promoting TTCR-C4 persistence. These findings suggest that AML drives a distinct form of T-cell dysfunction, highlight the need for targeted approaches that preserve T-cell fitness, ultimately improving the efficacy of cellular therapies for AML.

Neural progenitor cells (NPCs) hold immense potential as therapeutic candidates for neural regeneration, and materials-based strategies have emerged as attractive options for NPC expansion. However, maintaining NPC stemness has proven challenging in vitro, due to their propensity to form cell-dense neurospheres. While neurospheres promote cell-cell interactions required for NPC stem maintenance, they also restrict oxygen transport, leading to hypoxia and limited cell expansion. To overcome these limitations, we investigate two materials-based approaches to maintain NPC stemness: 1) physical matrix remodeling within a viscoelastic, stress-relaxing hydrogel and 2) matrix-induced N-cadherin-like signaling through a cell-instructive peptide. While viscoelasticity alone is sufficient to maintain NPC stemness compared to an elastic environment, NPCs still preferentially form neurospheres. The addition of N-cadherin-like peptides promotes a distributed culture of NPCs while maintaining their stemness through cadherin-mediated signaling, ultimately exhibiting improved long-term expansion and neural differentiation. Thus, our findings reveal matrix viscoelasticity and engineered N-cadherin-like interactions as having a synergistic effect on NPC expansion and differentiation within 3D matrices.

Satellite cells are muscle-resident stem cells that maintain and repair muscle. Increasing evidence supports the contributing role of satellite cells in Duchenne muscular dystrophy (DMD), a lethal degenerative muscle disease caused by loss of dystrophin. However, whether or not satellite cells exhibit dysfunction due to loss of dystrophin remains unresolved. Here, we used single-cell RNA-sequencing (scRNA-seq) to determine how dystrophin deficiency impacts the satellite cell transcriptome and cellular composition by comparing satellite cells from mdx and the more severe D2-mdx DMD mouse models. DMD satellite cells were disproportionally found within myogenic progenitor clusters and a previously uncharacterized DMD-enriched cluster. Despite exposure to different dystrophic environments, mdx and D2-mdx satellite cells exhibited overlapping dysregulation in gene expression and associated biological pathways. When comparing satellite stem cell versus myogenic progenitor populations, we identified unique dysfunctions between DMD and healthy satellite cells, including apoptotic cell death and senescence, respectively. Pseudotime analyses revealed differences in cell fate trajectories, indicating that DMD satellite cells are stalled in their differentiation capacity. In vivo regeneration assays confirmed that DMD satellite cells exhibit impaired myogenic gene expression and cell fate dynamics during regenerative myogenesis. These defects in differentiation capacity are accompanied by impaired senescence and autophagy dynamics. Finally, we demonstrate that inducing autophagy can rescue the differentiation of DMD progenitors. Our findings provide novel molecular evidence of satellite cell dysfunction in DMD, expanding on our understanding of their role in its pathology and suggesting pathways to target and enhance their regenerative capacity.

Hepatocellular carcinoma (HCC) is a leading cause of global cancer-associated mortality. Although various therapies have substantially ameliorated clinical outcome, patients invariably suffer from cancer relapse, highlighting the need for more optimized therapeutic strategies. Here, we report that deficiency of DNA methylcytosine dioxygenase TET2 sensitizes HCC cells to sorafenib and verteporfin treatments. Mechanistically, knockout of TET2 enhances the dephosphorylation of YAP Ser127, thus promoting its activity. RNA-seq analysis reveals that MC1R, a GPCR, is strikingly decreased upon TET2 deficiency. Furthermore, TET2 catalyzes demethylation of MC1R promoter to stimulate its transcription. MC1R subsequently boosts cAMP-PKA signaling to phosphorylate YAP Ser127 in both ligand dependent and independent manners. Importantly, deletion of MC1R accelerates tumor growth of HCC, which is reversed by the treatment of YAP-TEAD complex inhibitor verteporfin. Synergistic combination of MC1R expression driver vitamin C and its ligand α-MSH dramatically represses HCC growth. Notably, TET2-MC1R-YAP axis is evidenced in HCC specimens and plays a vital role in prognosis of HCC. Collectively, these findings not only elucidate a previously unidentified epigenetic regulatory mechanism of MC1R transcription and underscore the functional significance of MC1R signaling in tumorigenesis of HCC, but also provide potential targets and clinical strategies for HCC therapy.

Genetic predisposition plays a major role in the development of invasive pulmonary aspergillosis (IPA). The risk and course of IPA vary significantly among patients, yet continuously new genetic mechanisms that influence individual antifungal immune responses are being discovered. While genetic variability in IL-1 family cytokines is recognized as an important cause of disease susceptibility, it is unclear whether and how less-studied IL-1 family members, such as the IL-36 cytokine subfamily, are genetically regulated and influence the risk of infection.

Copper dysregulation has been linked to human health, disorders, and hematopoiesis. However, the underlying mechanisms remain elusive. Here, we demonstrate the pivotal role of dietary copper via the transporter Slc31a1(Ctr1) in copper homeostasis, but not cuproptosis, during postnatal hematopoiesis. Specifically, Slc31a1-mediated copper uptake sustains the differentiation and commitment of multipotent progenitors from short-term hematopoietic stem cells (HSCs). Using transcriptomic analyses, we reveal a disrupted differentiation program in hematopoietic stem and progenitor cells (HSPCs) in diet-induced copper-deficient mice or hematopoietic-specific Slc31a1 knockout (vKO) mice. Further, we show that Slc31a1 and copper are indispensable for sustaining mitochondrial activity via regulating Mtco1 and Mtco2 (subunits of Complex IV) within HSPCs. Notably, we show that the chemical compound elesclomol, also well-known as a potent cuproptosis agonist, significantly alleviates severe anemia and partially recovers HSPC mitochondrial function in vKO mice via its activity as a copper ionophore, but with no effect on cuproptosis. We thus renamed elesclomol as CupriActivitor1(CuA1), which is a more specific and descriptive term. These findings demonstrate the critical role and mechanism of copper, Slc31a1, and CuA1 in maintaining HSC homeostasis via modulation of mitochondrial energy metabolism. The study sheds light on the molecular basis of HSC fate decisions by copper or CuA1 and opens new avenues for the development of novel therapeutic strategies for copper-related disorders and blood diseases. Given the critical and multifaceted nature of copper, we propose establishing a novel interdisciplinary field termed "Cuprology". This discipline will advance our understanding of copper's roles in physiological and pathological processes.

Cancer stem cells (CSCs) are highly tumorigenic, self-renewable cells with a key role in tumor relapse, metastasis, and therapy resistance. Effective CSC-targeted therapies remain an unmet clinical need, strongly dependent on the selection of suitable targets and thorough validation of therapeutic agents. L1 cell adhesion molecule (L1CAM) is a targetable CSC-associated biomarker aberrantly expressed in various malignancies, including ovarian cancer (OC). 161Tb is attractive for clinical application because of its substantial emission of conversion electrons/Auger electrons as well as β- emission. Leveraging the high cytotoxicity of conversion electrons/Auger electrons, 161Tb is promising for radioimmunotherapy against radioresistant tumor cells such as CSCs. The aim of this study was to confirm the presence of L1CAM+/CD133+ ovarian CSCs in patient samples and preclinically investigate, in a tumor prevention mouse model, 161Tb-based anti-L1CAM radioimmunotherapy as a new therapeutic modality against CSCs compared with 177Lu-based anti-L1CAM radioimmunotherapy. Methods: L1CAM+/CD133+ CSCs were examined in OC samples by immunofluorescence. After radiolabeling anti-L1CAM DOTA-chCE7 with 177Lu or 161Tb and purification, we assessed radioimmunoconjugate quality by determining the radiochemical purity and the immunoreactive fraction. The internalized and membrane-bound fractions and the radiocytotoxicity of radiolabeled DOTA-chCE7 were evaluated with cell uptake and cell proliferation assays. Ovarian L1CAM+/CD133+ CSCs were sorted via fluorescence-activated cell sorting from OVCAR8 and SKOV3ip cells and inoculated into immunocompromised mice, who then received treatment with [177Lu]Lu-DOTA-chCE7 or [161Tb]Tb-DOTA-chCE7. Results: L1CAM+/CD133+ CSCs (0.3%-21%) were confirmed in samples from patients who were chemotherapy-naïve or had relapsed OC. [177Lu]Lu-DOTA-chCE7 and [161Tb]Tb-DOTA-chCE7 were produced with high radiochemical purity and retained 76%-96% immunoreactivity. Cell uptake after 15 h ranged from 50% to 75% for both radioimmunoconjugates. [161Tb]Tb-DOTA-chCE7 showed significantly increased cytotoxicity, eliminating all ovarian CSCs and tumor cells differentiated from the CSCs in vivo, compared with [177Lu]Lu-DOTA-chCE7 (3 tumors in OVCAR8 group and 1 tumor in SKOV3ip group). Follow-up tumor analysis confirmed that sorted ovarian L1CAM+/CD133+ CSCs regenerated the tumor heterogeneity in vivo. Conclusion: This work addresses the critical need for CSC-specific therapies in the clinics by establishing 161Tb-based anti-L1CAM radioimmunotherapy as a novel therapeutic modality against CSCs. We found that 161Tb-based anti-L1CAM radioimmunotherapy eliminated ovarian CSCs more efficiently than 177Lu-based anti-L1CAM radioimmunotherapy, emphasizing its promising therapeutic potential.

Ionizing radiation induces DNA double-strand breaks, which compromise genomic stability and trigger programmed cell death. The cell's differentiation state modulates DNA damage response (DDR) mechanisms, including DNA repair pathways and cell cycle regulation. The accumulation of p53-binding protein 1 (53BP1) at DSB sites serves as a reliable biomarker for such damage. Previously, we developed a fluorescent live-cell imaging system, termed "Focicle," which monitors 53BP1 foci dynamics and cell cycle phases, utilizing fluorescent ubiquitination-based cell cycle indicators (hCdt1 and hGmnn) in mouse cells. In the current study, to investigate the relationship between differentiation state and DDR activity, we generated Focicle-integrated human induced pluripotent stem cells and further differentiated them into neural progenitors and mature neurons using an optimized Focicle cassette adapted for human cell lines. Using laser microirradiation, we observed differentiation-dependent alterations in 53BP1 foci accumulation dynamics and cell cycle progression. The newly established Focicle system represents a valuable tool for elucidating DDR activity during organ development.

Neurotrophins, a type of growth factor, are key players in guiding mesenchymal stem cells (MSCs) towards becoming neurons. Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF) are well-known for promoting axon growth and pushing MSCs to resemble neural precursor cells. However, their short half-life has consistently generated issues related to their bioavailability, which is essential in determining the fate of stem cells during differentiation. Currently, we have developed a chitosan nanocarrier infused with NGF to enhance its availability in the stem cell niche. Additionally, it is combined with BDNF to formulate a neural induction medium that facilitates the neural differentiation of ADMSCs (Adipose-derived MSCs). The developed cells displayed a morphological appearance consistent with neurological lineage cells, confirmed by the presence of cellular markers linked to neural cells and synaptic connections. The statistical study confirmed that encapsulated NGF is a substantial neural inducer compared to neurotrophin employed alone in the induction medium. The chitosan nanocarrier exhibited advantageous NGF loading efficiency and release kinetics, making it suitable for enhancing neural differentiation. Consequently, the continuous release of NGF via chitosan nanocarriers has enhanced the promotion of neurogenic differentiation of ADMSCs and holds prospective applications in neural tissue engineering.

The complex microenvironment of bone defects, particularly enhanced oxidative stress, poses significant clinical challenges for bone repair. Furthermore, the inadequate regulation of the regenerative microenvironment by conventional biomaterials substantially compromises healing efficacy. Herein, we developed tannic acid (TA)‑magnesium (Mg) metal-phenolic network (MPN) coated mesoporous bioactive glass nanoparticles (MBGNs@TA-Mg) and integrated them into glycidyl methacrylate-grafted gelatin (Gel-GMA) to fabricate GM/MBTM hydrogels. In vitro studies demonstrated that GM/MBTM hydrogels with excellent cytocompatibility, effectively scavenging free radicals and reactive oxygen species (ROS), and significantly promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Meanwhile, the hydrogels displayed antimicrobial activity against Staphylococcus aureus (S. aureus). Therefore, this study establishes a novel strategy for designing multifunctional biomaterials capable of microenvironment modulation and enhanced bone regeneration.

Permethrin (PER) is a synthetic pyrethroid used in agricultural settings and public health. Previously, PER exposure has been linked with increased adipogenesis. The Wnt/β-catenin pathway is also associated with the regulation of adipogenic differentiation of mesenchymal stem cells (MSCs). This study evaluated the effect of acute PER exposure (24 hr) on the Wnt/β-catenin pathway and its impact on adipogenic commitment of MSC. It also assessed the cross-talk of Wnt/β-catenin signaling and autophagy in PER-mediated adipogenesis. Immunoblotting results showed PER inhibited the Wnt/β-catenin signaling. Nile red staining showed decreased adipogenesis upon PER pre-treatment in combination with lithium chloride compared to PER alone. Further, immunoblotting revealed lithium chloride-mediated activation of autophagy in MSCs. Nile red staining showed inhibition of adipogenic differentiation upon activation of the Wnt/β-catenin pathway and autophagy. Overall, the data showed that cross-talk of autophagy and the Wnt/β-catenin pathway negatively regulates adipogenic commitment of MSCs.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity, with chemoresistance is the greatest challenge in chemotherapeutic treatment. Stanniocalcin 1 (STC1) is correlated with tumor malignancy and chemoresistance in various cancers, but its role in OSCC paclitaxel (PTX) resistance remains elusive. This study aimed to clarify STC1's impact on OSCC PTX resistance and elucidate its underlying mechanism.

Neural stem cells (NSCs) are a promising therapy for central nervous system (CNS) disorders, yet post-transplant immune rejection critically compromises their survival and efficacy. In this study, we demonstrated the neuroinflammatory responses triggered by syngeneic, allogeneic, and xenogeneic NSCs transplantation, and evaluated the immunosuppressive effects of cyclosporine A (CyA) and methylprednisolone (MP) on graft rejection. Our findings revealed that xenogeneic NSCs transplantation induced infiltration of neutrophils (p < 0.0001), microglia/macrophages (p < 0.0001), CD4+ and CD8+ T cells (p < 0.0001), while allogeneic transplantation primarily triggered microglia/macrophages (p < 0.0005) recruitment. Both transplantation types caused a sharp decline in grafted cell numbers (p < 0.005). Combinatorial CyA and MP treatment significantly attenuated xenogeneic immune rejection and markedly increased surviving graft cells in the brain. Similarly, MP monotherapy effectively reduced allogeneic rejection and enhanced transplanted cell survival. Overall, allogeneic NSCs transplantation primarily triggers innate immunity, while xenogeneic transplantation causes both innate and adaptive immune responses. Accordingly, xenogeneic transplantation required combined CyA and MP therapy, whereas MP monotherapy mitigated rejection in allogeneic transplantation. Our findings may offer a strategy to mitigate transplantation rejection of allogeneic and xenogeneic NSCs in the brain, thereby optimizing the microenvironment for NSC-based therapies in preclinical and clinical applications for various CNS disorders.

Irradiation and 5-fluorouracil (5-FU) are widely utilized tools in hematopoietic research to generate myeloablation and assess blood recovery dynamics. A comprehensive understanding of their effects on the hematopoietic system is essential for optimizing therapeutic strategies, refining experimental models to modulate hematotoxicity, and interpreting research outcomes. Despite their widespread application, the long-term hematopoietic impacts of irradiation and 5-FU, particularly on hematopoietic stem cells (HSCs), remain incompletely characterized. In this study, we therefore examined the long-term effects of 2Gy medium-dose ionizing radiation (MDIR) and 150mg/kg 5-FU on HSCs and the hematopoietic system's resilience to subsequent cytotoxic stress in mice. Our findings demonstrate that MDIR, but not 5-FU, induces sustained impairments in HSC function and results in the selective depletion of MHC class II- HSCs - a subset characterized by high self-renewal potential and hypersensitivity to irradiation-induced ROS production. Furthermore, MDIR significantly compromised hematopoietic recovery following a subsequent 5-FU challenge, as evidenced by substantially reduced platelet and red blood cell (RBC) counts during the critical recovery phase. These findings highlight the distinct and persistent impacts of MDIR and 5-FU on HSCs and hematopoietic function, revealing crucial differences in their mechanisms of action and long-term consequences on the hematopoietic system.