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Atezolizumab plus bevacizumab and lenvatinib are standard treatments for unresectable hepatocellular carcinoma; however,tumor markers such as alpha-fetoprotein and des-gamma-carboxy prothrombin have a limited ability to reflect treatment responses. Circulating tumor cells are non-invasive biomarkers associated with cancer stemness and treatment resistance. We assessed circulating tumor cell subsets expressing cancer stem cell markers (CD90,epithelial cell adhesion molecule,CD133,vimentin) using multiparametric flow cytometry at early and maximal response phases in patients receiving atezolizumab plus bevacizumab or lenvatinib. Early decreases in CD90-positive circulating tumor cells after therapy initiation were associated with tumor shrinkage and longer progression-free survival in both groups,as well as prolonged overall survival in the atezolizumab plus bevacizumab group. At maximal response,changes in CD90-positive circulating tumor cells reflected tumor burden more accurately than alpha-fetoprotein or des-gamma-carboxy prothrombin. These findings highlight the potential of CD90-positive circulating tumor cells to become dynamic biomarkers in systemic therapy for unresectable hepatocellular carcinoma. View Publication

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the lack of ER,PR,and HER2 expression. Its aggressive behavior,high degree of tumor heterogeneity,and immunosuppressive tumor microenvironment (TME) are associated with poor clinical outcomes,rapid disease progression,and limited therapeutic options. Although chimeric antigen receptor (CAR)-engineered T cell therapy has shown certain promise,its applicability in TNBC is hindered by antigen escape,TME-mediated suppression,and the logistical constraints of autologous cell production. In this study,we employed hematopoietic stem and progenitor cell (HSPC) gene engineering and a feeder-free HSPC differentiation culture to generate allogeneic IL-15-enhanced,mesothelin-specific CAR-engineered invariant natural killer T ( Allo15 MCAR-NKT) cells. These cells demonstrated robust and multifaceted antitumor activity against TNBC,mediated by CAR- and NK receptor-dependent cytotoxicity,as well as selective targeting of CD1d + TME immunosuppressive cells through their TCR. In both orthotopic and metastatic TNBC xenograft models,Allo15 MCAR-NKT cells demonstrated potent antitumor activity,associated with robust effector and cytotoxic phenotypes,low exhaustion,and a favorable safety profile without inducing graft-versus-host disease. Together,these results support Allo15 MCAR-NKT cells as a next-generation,off-the-shelf immunotherapy with strong therapeutic potential for TNBC,particularly in the context of metastasis,immune evasion,and treatment resistance. The online version contains supplementary material available at 10.1186/s13045-025-01736-9. View Publication

The discovery and study of human neural stem cells has advanced our understanding of human neurogenesis,and the development of novel therapeutics based on neural cell replacement. Here,we describe methods to culture and cryopreserve human neural stem cells (hNSCs) for expansion and banking. Importantly,the protocols ensure that the multipotency of hNSCs is preserved to enable differentiation to neurons and supporting neuroglia. View Publication

BACKGROUND Longitudinal studies have verified the pivotal role of mesenchymal stem/stromal cells (MSCs) in the bone marrow microenvironment for hematopoiesis and coordinate contribution to leukemia pathogenesis. However,the precise characteristics and alternation of MSCs during acquired aplastic anemia (AA) remain obscure. METHODS In this study,we originally collected samples from both healthy donors (HD) and AA patients to dissect the hematological changes. To systematically evaluate the biological defects of AA-derived MSCs (AA-MSCs),we analyzed alterations in cellular morphology,immunophenotype,multi-lineage differentiation,cell migration,cellular apoptosis,and chromosome karyocyte,together with the immunosuppressive effect on the activation and differentiation of lymphocytes. With the aid of whole genome sequencing and bioinformatic analysis,we try to compare the differences between AA-MSCs and HD-derived MSCs (HD-MSCs) upon the molecular genetics,especially the immune-associated gene expression pattern. In addition,the efficacy of umbilical cord-derived MSC (UC-MSC) transplantation on AA mice was evaluated by utilizing survivorship curve,histologic sections,and blood cell analyses. RESULTS In coincidence with the current reports,AA patients showed abnormal subsets of lymphocytes and higher contents of proinflammatory cytokines. Although with similar immunophenotype and chromosome karyotype to HD-MSCs,AA-MSCs showed distinguishable morphology and multiple distinct characteristics including genetic properties. In addition,the immunosuppressive effect on lymphocytes was significantly impaired in AA-MSCs. What is more,the cardinal symptoms of AA mice were largely rescued by systemic transplantation of UC-MSCs. CONCLUSIONS Herein,we systematically investigated the signatures and efficacy of MSCs to dissect the alterations occurred in AA both at the cellular and molecular levels. Different from HD-MSCs,AA-MSCs exhibited multifaceted defects in biological characteristics and alterative molecular genetics in the whole genome. Our findings have provided systematic and overwhelming new evidence for the defects of AA-MSCs,together with effectiveness assessments of UC-MSCs on AA as well. View Publication

Understanding the root causes of autoimmune diseases is hampered by the inability to access relevant human tissues and identify the time of disease onset. To examine the interaction of immune cells and their cellular targets in type 1 diabetes,we differentiated human induced pluripotent stem cells into pancreatic endocrine cells,including $\beta$ cells. Here,we describe an in vitro platform that models features of human type 1 diabetes using stress-induced patient-derived endocrine cells and autologous immune cells. We demonstrate a cell-type-specific response by autologous immune cells against induced pluripotent stem cell-derived $\beta$ cells,along with a reduced effect on $\alpha$ cells. This approach represents a path to developing disease models that use patient-derived cells to predict the outcome of an autoimmune response. View Publication

The formulations of peptide-based antitumour vaccines being tested in clinical studies are generally associated with weak potency. Here,we show that pharmacokinetically tuning the responses of peptide vaccines by fusing the peptide epitopes to carrier proteins optimizes vaccine immunogenicity in mice. In particular,we show in immunized mice that the carrier protein transthyretin simultaneously optimizes three factors: efficient antigen uptake in draining lymphatics from the site of injection,protection of antigen payloads from proteolytic degradation and reduction of antigen presentation in uninflamed distal lymphoid organs. Optimizing these factors increases vaccine immunogenicity by up to 90-fold and maximizes the responses to viral antigens,tumour-associated antigens,oncofetal antigens and shared neoantigens. Protein-peptide epitope fusions represent a facile and generalizable strategy for enhancing the T-cell responses elicited by subunit vaccines. View Publication

Severe acute respiratory syndrome (SARS) coronavirus (CoV) envelope (E) protein is a transmembrane protein. Several subcellular locations and topological conformations of E protein have been proposed. To identify the correct ones,polyclonal and monoclonal antibodies specific for the amino or the carboxy terminus of E protein,respectively,were generated. E protein was mainly found in the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) of cells transfected with a plasmid encoding E protein or infected with SARS-CoV. No evidence of E protein presence in the plasma membrane was found by using immunofluorescence,immunoelectron microscopy and cell surface protein labeling. In addition,measurement of plasma membrane voltage gated ion channel activity by whole-cell patch clamp suggested that E protein was not present in the plasma membrane. A topological conformation in which SARS-CoV E protein amino terminus is oriented towards the lumen of intracellular membranes and carboxy terminus faces cell cytoplasm is proposed. View Publication

Aim of work was to locate a simple,reproducible protocol for uniform seeding and optimal cellularization of biodegradable patch minimizing the risk of structural damages of patch and its contamination in long-term culture. Two seeding procedures are exploited,namely static seeding procedures on biodegradable and biocompatible patches incubated as free floating (floating conditions) or supported by CellCrownTM insert (fixed conditions) and engineered by porcine bone marrow MSCs (p-MSCs). Scaffold prototypes having specific structural features with regard to pore size,pore orientation,porosity,and pore distribution were produced using two different techniques,such as temperature-induced precipitation method and electrospinning technology. The investigation on different prototypes allowed achieving several implementations in terms of cell distribution uniformity,seeding efficiency,and cellularization timing. The cell seeding protocol in stating conditions demonstrated to be the most suitable method,as these conditions successfully improved the cellularization of polymeric patches. Furthermore,the investigation provided interesting information on patches' stability in physiological simulating experimental conditions. Considering the in vitro results,it can be stated that the in vitro protocol proposed for patches cellularization is suitable to achieve homogeneous and complete cellularizations of patch. Moreover,the protocol turned out to be simple,repeatable,and reproducible. View Publication

Human pluripotent stem cells including induced pluripotent stem cells (iPSCs) and embryonic stem cells hold great promise for cell-based therapies,but safety concerns that complicate consideration for routine clinical use remain. Installing a safety switch" based on the inducible caspase-9 (iCASP9) suicide gene system should offer added control over undesirable cell replication or activity. Previous studies utilized lentiviral vectors to integrate the iCASP9 system into T cells and iPSCs. This method results in random genomic insertion of the suicide switch and inefficient killing of the cells after the switch is "turned on" with a small molecule (eg AP1903). To improve the safety and efficiency of the iCASP9 system for use in iPSC-based therapy we precisely installed the system into a genomic safe harbor the AAVS1 locus in the PPP1R12C gene. We then evaluated the efficiencies of different promoters to drive iCASP9 expression in human iPSCs. We report that the commonly used EF1$\alpha$ promoter is silenced in iPSCs and that the endogenous promoter of the PPP1R12C gene is not strong enough to drive high levels of iCASP9 expression. However the CAG promoter induces strong and stable iCASP9 expression in iPSCs and activation of this system with AP1903 leads to rapid killing and complete elimination of iPSCs and their derivatives including MSCs and chondrocytes in vitro. Furthermore iPSC-derived teratomas shrank dramatically or were completely eliminated after administration of AP1903 in mice. Our data suggest significant improvements on existing iCASP9 suicide switch technologies and may serve as a guide to other groups seeking to improve the safety of stem cell-based therapies." View Publication

INTRODUCTION The stem cell portion of the dental pulp derived cultures (DPSCs) showed a higher resistance to cytotoxic effect of restorative dental materials compared to pulpal fibroblasts (DPFs). Here,we aimed to compare the expression of some drug resistant genes between these cells. METHODS AND MATERIALS To separate DPSCs from DPFs,we used magnetic cell sorting technique based on CD146 expression. To assess the stem cell properties,the positive and negative portions underwent colony forming assays and were induced to be differentiated into the adipocytes,osteoblasts,hepatocytes,and neural cells. Cell surface antigen panels were checked using immune fluorescence and flow-cytometry techniques. The mRNA expression of 14 ABC transporters including ABCA2,ABCB1,ABCB11,ABCC1,ABCC2,ABCC3,ABCC4,ABCC5-2,ABCC5-4,ABCC5-13,ABCC6,ABCC10,ABCC11,and ABCG2 genes was assessed,using quantitative RT-PCR technique. RESULTS Only the CD146 positive portion could be differentiated into the desired fates,and they formed higher colonies (16.7 ± 3.32 vs. 1.7 ± 1.67,p {\textless} .001). The cell surface antigen panels were the same,except for CD146 and STRO-1 markers which were expressed only in the positive portion. Among the ABC transporter genes studied,the positive portion showed a higher expression (approximately two-fold) of ABCA2,ABCC5-13,and ABCC5-2 genes. CONCLUSION Dental pulp stem cells which can be separated from dental pulp fibroblasts based on CD146 expression,express higher levels of some drug resistance genes which probably accounts for their features of more resistance to cytotoxic effects of some dental materials. This needs to be more validated in future. View Publication

Increasing occurrence of moderate to severe intrauterine adhesion (IUA) is seriously affecting the quality of human life. The aim of the study was to establish IUA models in nonhuman primates and to explore the dual repair effects of human umbilical cord-derived mesenchymal stem cells (huMSCs) loaded on autocrosslinked hyaluronic acid gel (HA-GEL) on endometrial damage and adhesion. Here,we recorded the menstrual cycle data in detail with uterine cavities observed and endometrial tissues detected after intervention,and the thicker endometria,decreased amount of fibrotic formation,increased number of endometrium glands,etc.,suggested that both HA-GEL and huMSC/HA-GEL complexes could partially repair IUA caused by mechanical injury,but huMSC/HA-GEL complex transplantation had notable dual repair effects: a reliable antiadhesion property and the promotion of endometrial regeneration. View Publication

Malignant cells have a higher nicotinamide adenine dinucleotide (NAD(+)) turnover rate than normal cells,making this biosynthetic pathway an attractive target for cancer treatment. Here we investigated the biologic role of a rate-limiting enzyme involved in NAD(+) synthesis,Nampt,in multiple myeloma (MM). Nampt-specific chemical inhibitor FK866 triggered cytotoxicity in MM cell lines and patient MM cells,but not normal donor as well as MM patients PBMCs. Importantly,FK866 in a dose-dependent fashion triggered cytotoxicity in MM cells resistant to conventional and novel anti-MM therapies and overcomes the protective effects of cytokines (IL-6,IGF-1) and bone marrow stromal cells. Nampt knockdown by RNAi confirmed its pivotal role in maintenance of both MM cell viability and intracellular NAD(+) stores. Interestingly,cytotoxicity of FK866 triggered autophagy,but not apoptosis. A transcriptional-dependent (TFEB) and independent (PI3K/mTORC1) activation of autophagy mediated FK866 MM cytotoxicity. Finally,FK866 demonstrated significant anti-MM activity in a xenograft-murine MM model,associated with down-regulation of ERK1/2 phosphorylation and proteolytic cleavage of LC3 in tumor cells. Our data therefore define a key role of Nampt in MM biology,providing the basis for a novel targeted therapeutic approach. View Publication