The generation of loss-of-function alleles in human pluripotent stem cells (hPSCs) is used to interrogate gene function and validate reagents; however, identifying clones harboring true loss-of-function alleles remains inefficient. To address this, we present BOLT (barcoded oligos for loss-of-function targeting), a streamlined protocol that simplifies the screening process, facilitating rapid validation of loss-of-function mutations. We describe steps for designing editing tools, nucleofection, and clonal density plating. We then detail procedures for bridging PCR, isolating clones derived from a single hPSC, single-clone screening, and Sanger barcode detection. For complete details on the use and execution of this protocol, please refer to Matera et al.1.

Acute myeloid leukemia (AML) is a heterogeneous malignancy specified by clonal proliferation of hematopoietic stem cells. This study identifies novel therapeutics for AML by integrating differential gene expression (DEG) and survival analyses. Publicly available GEO microarray datasets were analyzed, including data from 615 AML patients and 22 healthy controls. Multivariate Cox regression identified hazardous genes impacting survival. Protein-protein interaction networks using CytoScape revealed hub genes such as CCT5, ZBTB16, APP, and PTPN6. Functional enrichment revealed key AML-related pathways, such as PI3K/Akt and NF-kappaB signaling. Drug repurposing using the LINCS L1000CDS2 database highlighted potential therapeutics, including 16-Hydroxytriptolide and Tryptosthin AG-1478, with roles in reversing hazardous gene expression patterns. Additional candidates such as Vemurafenib, Parthenolide and Wortmannin, demonstrated promise as targeted agents. These findings underscore the potential of integrating bioinformatics and drug discovery to identify precision medicine in AML. Further studies are warranted to validate these targets and explore their clinical utility.

Osteosarcoma (OS), chondrosarcoma (CHS), and Ewing sarcoma (EwS) are the most common primary bone cancers (BCs). Among primary malignant tumors of the bones, OS is the most common, mainly affecting young people (4.8 per 1,000,000). The treatment of bone cancer (BC) is challenging for current medicine owing to its substantial incidence and the vast heterogeneity of malignant lesions within bone tissue. Due to the limitations of current therapies, researchers developed new strategies to treat BC. Exosomes (EXOs) play a crucial role in the development, progression, metastasis, and drug delivery of BCs, such as OS, EwS, and CHS. Hierarchical translation via tissue-specific reactions and cell-specific molecular signaling pathways accounts for the various therapeutic effects of EXOs produced from stem cells. The aim of this review is to highlight the critical role of EXOs derived from multiple cells, such as mesenchymal stem cells, immune cells, and tumor cells, in BCs, including OS, CHS, and EwS. Additionally, we provide a concise overview of how tumor-derived EXOs induce BCs. To lessen the adverse effects of EXOs on patients with BC and to provide more effective and focused treatments, it is necessary to understand these pathways. Moreover, we reviewed the potential of using EXOs as drug delivery systems for the treatment of BCs. Finally, we discussed the pros and cons of this therapeutic approach for BCs.

Accurately detecting cell secretions in complex microenvironments is crucial for understanding cellular communication, disease progression, and therapeutic responses. Traditional methods, such as ELISA, provide limited insight into the spatiotemporal dynamics of secretions, often requiring invasive or endpoint analyses. To address these challenges, we have functionalized the CellStudio platform-previously developed to integrate cell adhesion areas and microbeads patterns-with a novel self-reporting structure-switching signaling aptamer (SSSA) for vascular endothelial growth factor (VEGF) detection. This functionalization enables in situ single-step, real-time quantification of VEGF secretion from hundreds of live mesenchymal stem cell (MSC) clusters over a 24-h period, using standard fluorescence microscopy without the need for cell fixation. The spatial resolution provided by this system enables precise, localized monitoring of secretion events without the need to add extra reagents. This user-friendly platform is easily adaptable to conventional laboratory workflows, offering a versatile tool with potential for studying cellular behavior in real time with minimal technical barriers.

Multiple sclerosis (MS) leads to histological changes, which in turn result in functional deficits. Studies have reported that bone marrow mesenchymal stem cells (BMSCs) have therapeutic benefits in MS. In addition, sleep deprivation affects therapeutic potential of BMSCs. This research aimed to evaluate the effects of BMSCs compared to sleep-deprived BMSCs (SD-BMSCs) on the motor behavior and histological recovery in MS. The mice were divided into four groups receiving cuprizone for five weeks, with or without intranasal administration of cells (BMSCs or SD-BMSCs) after five weeks. After a two-week recovery period, balance beam and pole tests were performed to assess motor performance, followed by estimation of histological parameters using stereological methods. Mice that received BMSCs scored significantly better than the SD-BMSC group in balance beam and pole tests (P < 0.05). The total cerebellar volume, cortex, and white matter volumes, as well as the total number of Purkinje cells in mice that received BMSCs, were significantly higher compared to those in the SD-BMSC group (P < 0.05). Intranasal administration of BMSCs leads to significantly improved motor behavior and histological regeneration of the cerebellum, including cortex and white matter as well as Purkinje cells compared to SD-BMSCs in a cuprizone model of multiple sclerosis.

Photobiomodulation (PBM), which stimulates cellular functions through light emission, has recently attracted significant attention in regenerative medicine. This study explores the effects of PBM with two wavelengths on human adipose-derived stem cells (ADSCs) and the characteristics of their secreted exosomes in vitro. ADSCs were isolated from human adipose tissue and treated with PBM with 650 nm and 810 nm wavelengths. The therapeutic effects, including cell viability, proliferation, and exosome characteristics, were evaluated using MTT assays, scanning electron microscopy (SEM), BCA assays to determine exosomal protein concentration, and Dynamic Light Scattering (DLS) to assess exosome size distribution. The results indicated that PBM at both wavelengths significantly enhanced ADSCs viability and proliferation, as confirmed by the MTT assay (p = 0.008 for 650 nm laser and p = 0.007 for 810 nm laser). Additionally, exosome analysis revealed that exosomes from PBM-treated groups exhibited a higher protein concentration and an average size of around 86 nm, as measured by BCA and DLS. We concluded that PBM with 650 nm and 810 nm wavelengths individually significantly enhances the proliferation and survival of ADSCs and improves exosome quality in vitro. The treatment increased exosomal protein concentration and improved size distribution, reflecting enhanced cell function. These findings suggest that PBM could be a promising stimulatory approach for improving ADSC-based therapies in regenerative medicine, particularly for tissue repair and chronic wound healing. Further research is needed to explore the molecular mechanisms and clinical applications of ADSCs under the influence of PBM.

Periodontal and craniofacial regeneration presents significant challenges owing to the complex tissue architecture, inadequate vascularization, and diminished stem cell populations within damaged tissues. Traditionally, autologous bone grafts or alternative bone substitute materials have been employed to address these conditions; however, these approaches are constrained by donor site morbidity, limited availability, and suboptimal regenerative efficacy. The advancement of mesenchymal stem/stromal cell (MSC) biology has accelerated the development of cell-based therapies in modern dentistry, which now focuses on biologically driven approaches to regenerate tissues. MSC-based therapies currently under investigation, both preclinically and clinically, show promise for improving tissue integration and healing processes of both soft and hard tissues, attributable to their multipotent nature, immunomodulatory properties, and paracrine signaling capabilities. Nevertheless, obstacles persist, including inconsistent standardization, limited scalability, regulatory hurdles, a paucity of controlled studies, and restricted biomaterial options. This review evaluates MSC-based treatments for periodontal and craniofacial reconstruction by discussing recent research findings and existing obstacles. This review also examines future prospects, such as advanced biofabrication methods, including 3D printing and bioprinting, which have the potential to improve personalized cell therapy for periodontal and craniofacial regeneration.

The extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSC-EVs) exhibit immunoregulatory functions dependent on their parent cells. MSC-EVs are promising candidates for treating neuroinflammation in neurological diseases due to their acellular nature and their ability to reach the central nervous system. However, the conditions of MSCs for producing EVs with the highest anti-inflammatory efficacy are still unknown. Therefore, the first objective was to study the characteristics of the EVs produced by MSCs cultured in different conditions. The second objective was to evaluate the in vitro anti-inflammatory properties of those EVs in feline stimulated mixed glia. Umbilical cord-derived MSCs were treated with serum-free (SF) media, inflammatory (IF) media, or media supplemented with 5% EV-depleted fetal bovine serum (FBS). The isolated MSC-EVs were characterized by particle size and yield, and their anti-inflammatory ability was evaluated in lipopolysaccharide (LPS) stimulated feline mixed glia. All EV isolates were <160 nm, and the primary mixed glia consisted of microglia, astrocytes, neurons, and endothelial cells. Our results indicate that IF-EVs statistically significantly decreased the production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) and downregulated the transcription of the, nuclear factor kappa B p65 subunit in inflammatory mixed glia after 48 hours. In addition, SF- and FBS-EVs significantly reduced in vitro the secretion of IL-6 after 48 hours, but only SF-EVs achieved a significant effect on inhibiting the expression of p65 at 48 hours. Moreover, messenger RNA (mRNA) levels of inducible nitric oxide synthase (iNOS) were significantly decreased following treatment with SF-EV for 24 hours. This study demonstrates that MSC culture conditions affect the therapeutic potential of the secreted EVs in feline mixed glia.

This study aims to investigate the expression profile and clinical significance of the Troponin T Type 1 (TNNT1) gene across various digestive system tumors. By elucidating the role of TNNT1 in tumor progression, we hope to establish its potential as a prognostic biomarker and therapeutic target.

Ovarian cancer remains one of the most aggressive cancers, and resistance to Poly (ADP-ribose) Polymerase inhibitors (PARPi) poses a major therapeutic challenge. SIRT5, a NAD + -dependent desuccinylase, plays a crucial role in regulating fatty acid metabolism, which is often reprogrammed in cancer cells to promote drug resistance. This study aimed to investigate the potential of polydopamine (PDA)-polymerized antioxidant nanozyme-loaded SIRT5-modified human umbilical cord mesenchymal stem cells (hUCMSCs) to overcome PARPi resistance in ovarian cancer. We employed multi-omics approaches, including transcriptomics, metabolomics, and proteomics, to identify key molecular pathways associated with resistance mechanisms. High-throughput sequencing and metabolic profiling revealed that SIRT5 modifies fatty acid β-oxidation and regulates the desuccinylation of Enoyl-CoA Hydratase (ECHA), a key enzyme involved in this process. In vitro and in vivo experiments demonstrated that nanozyme-engineered hUCMSCs effectively enhanced PARPi resistance by promoting fatty acid metabolism and desuccinylation. These findings suggest that SIRT5-modified hUCMSCs loaded with antioxidant nanozymes offer a promising therapeutic strategy to combat PARPi resistance in ovarian cancer. The study provides new insights into overcoming drug resistance through metabolic reprogramming and enhances the potential of engineered stem cells in cancer therapy.

M2 macrophages are known to be involved in tumorigenesis. However, the mechanism by which they promote tumor progression in endometrial cancer (EC) remains largely unknown. Kynureninase (KYNU) has been found to be associated with the progression of various tumors, but research on endometrium is limited to embryo transfer. Therefore, a better understanding of KYNU as a potential therapeutic target in EC treatment is needed. This study aimed to elucidate the mechanism by which M2 macrophage-secreted KYNU influences the malignant biological and stemness remodeling of EC via the SOD2-mtROS-ERO1α and endoplasmic reticulum unfolded protein response (UPRER) pathway.

Intrinsically resistant glioma stem cells (GSCs) in the setting of a highly immunosuppressive tumor microenvironment (TME) remain the most predominant phenomenon leading to unfavorable therapeutic outcomes in glioblastoma (GBM). Hence there is an unmet need for novel anti-GBM therapeutic paradigms that can effectively target GSCs while simultaneously reprogramming the TME.

Surgical treatment alone is not effective in addressing delayed fracture healing (DFH). This study nvestigates the molecular mechanism underlying fracture healing to identify improved therapeutic strategies.

While the recent FDA-approved antiandrogen enzalutamide (Enz) might prolong the survival of castration-resistant prostate cancer (CRPC) patients by an additional 4.8 months, most patients eventually might still develop Enz resistance within 6-12 months. Although few genes have been linked to Enz resistance in prostate cancer (PCa), the detailed mechanism(s) are still underinvestigated. Here, we found that Enz might function by altering androgen receptor(AR)-mediated CDR1/circCDR1-AS/miR-1290/BMP4 signaling to modulate PCa stem cells (CSCs) to increase Enz resistance. Mechanistic analysis revealed that Enz/AR signaling can transcriptionally regulate CDR1 expression by reducing binding to androgen response elements (AREs) on the CDR1 5' promoter to alter circCDR1-AS expression. Enz/AR/CDR1/circCDR1-AS signaling might then increase BMP4 expression by altering miR-1290 expression, which involves direct binding to the 3' UTR of BMP4 mRNA. Preclinical studies using a CWR22Rv1 xenograft mouse model and integrative analysis of GEO cohort data further demonstrated that targeting this newly identified Enz/AR/CDR1/circCDR1-AS/miR-1290/BMP4 signaling pathway with miR-1290, circCDR1-AS-shRNA, or BMP4-shRNA may help develop novel therapies to combat Enz resistance at the later stage of CRPC.

This study aimed to evaluate the therapeutic effects of adipose tissue-derived stem cell exosomes (ADSCE) in the management of stress urinary incontinence (SUI) using vaginal dilatation-induced acute and chronic SUI models in Sprague-Dawley rats. The model for acute and chronic SUI was established via single and repeated vaginal dilatation, respectively. Mesenchymal cells, mesenchymal stem cells, and exosomes were isolated. Flow cytometry confirmed the mesenchymal identity of isolated stem cells, while exosomal markers (CD9, CD63, CD81) validated successful exosome isolation. Transmission electron microscopy revealed the characteristic nanoscale morphology of exosomes, further supported by energy-dispersive X-ray analysis. Rats were treated with the exosomes locally and vaginally. Therapeutic efficacy was assessed through urodynamic and histopathological analyses. Abdominal leak point pressure (ALPP) was significantly reduced in both acute and chronic SUI models compared to controls (P < 0.0001). ADSCE therapy significantly increased ALPP, with systemic administration demonstrating superior efficacy over local treatment (P < 0.05). Histopathological examination indicated substantial sphincter muscle thinning, edema, and fibrosis in untreated models, while exosome therapy mitigated these pathological changes. Masson's Trichrome staining revealed significant preservation of urethral sphincter thickness in treated groups (P < 0.01), with systemic therapy yielding moderate improvements over local administration. Furthermore, exosome therapy markedly reduced collagen deposition, particularly in systemic treatment groups, suggesting an antifibrotic effect and enhanced tissue remodeling. These findings indicate that ADSCE effectively restores urethral function and mitigates pathological alterations in SUI. Systemic administration demonstrates superior therapeutic potential, highlighting ADSCE therapy as a promising regenerative strategy for SUI management.

For many corneal diseases, transplantation is the gold standard for curative treatment and restoration of vision. Penetrating keratoplasty (PKP), performed by Zirm in 1905, was the first successful corneal transplantation procedure. Since then, relentless advancement in the field has occurred, most importantly with the development of deep anterior lamellar keratoplasty (DALK), Descemet stripping automated endothelial keratoplasty (DSAEK) and Descemet membrane endothelial keratoplasty (DMEK), which have been rapidly increasing in usage and are poised to take over PKP in prevalence and effectiveness in treating specific stromal and endothelial pathologies. The biggest issues currently facing this field are the lack of availability of donor corneas and lack of accessibility of the procedure to many areas of the world. Recent and future advancements are focused on substitutes to increase the amount of graft material for use and technological developments to streamline keratoplasty techniques, making them more effective, easier to perform and associated with less complications. Bio-engineered corneas, cell-based therapies and regenerative medicine can create grafts through various mechanisms: acellular, synthetic scaffolds and medical therapies to promote endogenous cell regeneration or exogenous cultivation of corneal tissues from stem-cells. Keratoplasty has also been refined by the introduction of femtosecond laser (FSL), which when combined with intra-operative optical coherence tomography (iOCT) allows for finer cuts and novel techniques which can improve the outcomes from PKP, DALK and DMEK.

Aspergillus fumigatus is a saprophytic fungus dwelling in soil and on decaying plant material, but also an opportunistic pathogen in immunocompromised patients. In its environmental niche, A. fumigatus faces competition from other microorganisms including bacteria. Here, we describe the discovery of the first secreted antibacterial protein in A. fumigatus. We identify a secreted fungal endopeptidase, designated CwhA, that cleaves peptidoglycan of Gram-positive bacteria at specific residues within the peptidoglycan stem peptide. Cleavage leads to bacterial lysis and the release of peptidoglycan cleavage products. Expression of cwhA is induced by the presence of bacteria. Furthermore, CwhA is highly abundant in murine lungs during invasive pulmonary aspergillosis and peptidoglycan cleavage products generated by CwhA stimulate cytokine production of human immune cells in vitro. Although CwhA does not affect human cells directly, this novel player in fungal-bacterial interactions could affect A. fumigatus infections by inhibiting Gram-positive bacteria in its vicinity, and possibly modulate the immune system.

The majority of cancer cells experience replication stress, which ultimately causes them to enter mitosis with underreplicated DNA. To alleviate the consequences of replication stress, cells utilize a mechanism known as MiDAS that functions to complete synthesis of underreplicated DNA in early mitosis. This process is considered an Achilles heel for highly replicative cancers. In this study, we show that human TopBP1 localizes to sites of underreplicated DNA marked by FANCD2 and promotes MiDAS through recruitment of the nuclease scaffold protein SLX4. Additionally, we demonstrate that the recruitment of SLX4 to TopBP1 foci in mitosis depends on TopBP1-K704, SLX4-T1260, and several SUMO-interaction motifs in SLX4. Lastly, we show that the recruitment of SLX4 to TopBP1 foci in mitosis is important to prevent transmission of DNA damage to daughter cells. Based on this, we hypothesize that targeting the TopBP1-SLX4 interaction in mitosis may be a potential strategy for anti-cancer therapy.

Pancreatic cancer stem cells (PCSCs) are a small population of cells in tumours that exhibit enhanced self-renewal and differentiation capabilities. CSCs proactively remodel the tumour microenvironment to maintain CSC stemness, which contributes to chemotherapy resistance. Compared with targeting PCSCs themselves, targeting the PCSC niche may be a novel strategy for pancreatic cancer (PC) therapy. Here, we found that DSG2, a member of the desmosomal cadherin family, is highly expressed in PCSCs. DSG2 upregulation is correlated with adverse outcomes in PC patients. DSG2 knockdown suppressed IL-4 and GM-CSF expression, which promoted the enrichment of tumour-associated macrophages to establish a supportive PCSC niche. Furthermore, we found that the IL-8/CXCR2 axis interacts with DSG2 to promote PCSC stemness and gemcitabine resistance by activating the Wnt/β-catenin pathway. These findings highlight the novel regulatory mechanism of DSG2 in PC, providing new targets for the development of therapeutics targeting PCSC niches.