技术资料

Pancreatic β cells derived from human induced pluripotent stem cells (hiPSCs) represent a promising therapeutic avenue in regenerative medicine for diabetes treatment. However,current differentiation protocols lack the specificity and efficiency required to reliably produce fully functional β cells,limiting their clinical applicability. Epigenetic barriers,such as histone modifications,may hinder proper differentiation and the acquisition of essential maturation markers in these cells. Methods: hiPSCs were cultured under feeder-free conditions and subjected to lentiviral transduction with shRNA constructs to silence KDM4A. Differentiation into pancreatic β-like cells was performed using stepwise protocols,with or without doxycycline supplementation,to evaluate the effect of KDM4A suppression. Gene expression was quantified by RT-qPCR,protein expression was assessed by western blotting and immunofluorescence,and functional insulin release was determined by glucose-stimulated insulin secretion (GSIS) assays. Statistical analysis was conducted using unpaired two-tailed Student’s t-tests,with significance set at p < 0.05. Results: A reduction in pancreatic development proteins was observed in the different differentiation states evaluated,after blocking KDM4A expression. Knockdown of KDM4A significantly reduced the expression of pancreatic β-cell genes,such as PDX1,Nkx6.1,and Ins,by 50% compared to WT iPSCs differentiated under the same conditions. Similarly,glucose-stimulated insulin secretion was reduced by approximately 80% in KDM4A-deficient β-like cells. Conclusions: These results emphasize the critical role of histone demethylation in hiPSC differentiation toward β cells. Our findings identify KDM4A as a key epigenetic regulator,suggesting that its modulation could enhance the generation of functional β cells for regenerative medicine in diabetes. View Publication

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by desmoplastic stroma,immunosuppressive tumor microenvironment (TME),and resistance to standard therapies. Natural killer (NK) cell-based immunotherapies have shown limited efficacy due to impaired persistence,infiltration,and function in PDAC. Methods: We established a direct reprogramming strategy to generate cytotoxic NK cells (1 F-NKs) by targeting BCL11B,a transcription factor essential for T cell lineage commitment,using shRNA or CRISPR/Cas9 in peripheral blood mononuclear cells (PBMCs). A genome-wide CRISPR/Cas9 screen identified tumor-intrinsic modulators of NK resistance. Functional and in vivo studies assesses the efficacy of 1 F-NKs alone and in combination with mesothelin (MSLN)-CAR engineering and PKMYT1 inhibition. Results: BCL11B depletion enabled the generation of CD56brightCD16bright 1 F-NKs with potent cytotoxicity and elevated NKG2D and CX3CR1 expression. Site-specific integration of a mesothelin (MSLN)-CAR into BCL11B locus generated MSLN-1 F-NKs with stable antigen specific activity. A genome-wide screen identified PKMYT1 as a modulator of tumor resistance to NK cell-mediated killing; its inhibition by RP6306 upregulated NKG2D ligands (MICA/B) and CX3CL1,sensitizing PDACs to 1 F-NK cytotoxicity. In PDAC xenograft models,1 F-NKs alone or combined with CAR engineering and RP6306 significantly reduced tumor growth and prolonged survival. Notably,this triple combination elicited a synergistic antitumor effect,outperforming each monotherapy or dual combination. Conclusions: This study presents a synergistic immunotherapy platform that integrates NK reprogramming,CAR engineering,and tumor sensitization. The combinatorial approach significantly enhances antitumor efficacy in PDAC and offers a promising strategy for overcoming immune resistance in solid tumors. View Publication

Cancer stem cells were prominent responsible for cancer initiation,metastasis,and invasion as well as therapeutic resistance in colorectal cancer (CRC). The extracellular axon guidance factor netrin-1 has been found to be overexpressed in several malignant cancers such as glioma,lung cancers,and colorectal cancer. However,the role of netrin-1 on cancer stemness in CRC remains unveiled. Our study revealed high expression of netrin-1 in colorectal cancer tissues and its ability to promote cancer stemness by interacting with receptors UNC5B and neogenin on murine colorectal cancer cell. Mechanistically,the netrin-1-UNC5B/neogenin axis activates the downstream NF-κB and ERK1/2 signaling pathways,reinforcing the stemness properties of tumor cells,and further exacerbating tumor progression. Clinically,netrin-1 expression associated with poor survival and high CD133 expression in patients with CRC. Taken together,these results suggest that netrin-1 blockade could be a compelling therapeutic strategy to improve the poor outcomes and trigger cancer stemness inhibition in CRC treatment. View Publication

Chemotherapy-induced peripheral neuropathy (CIPN) affects up to two-thirds of cancer patients undergoing cytotoxic chemotherapy. Here,we used human iPSC-derived sensory neurons (iPSC-DSN) to model CIPN in vitro. Administration of various chemotherapeutic agents (i.e.,paclitaxel,vincristine,bortezomib and cisplatin) at clinically applicable concentrations resulted in reduced cell viability,axonal degeneration,electrophysiological dysfunction and increased levels of phosphorylated c-Jun in iPSC-DSN. Transcriptomic analyses revealed that the upregulation of c-Jun strongly correlated with the expression of genes of neuronal injury,apoptosis and inflammatory signatures. To test whether c-Jun plays a central role in the development of CIPN,we applied the small molecule inhibitor of the Jun N-terminal kinase,SP600125,to iPSC-DSN treated with neurotoxic chemotherapy. c-Jun inhibition prevented chemotherapy-induced neurotoxicity by preserving cell viability,axonal integrity and electrophysiological function of iPSC-DSN. These findings identify c-Jun as a key mediator of CIPN pathophysiology across multiple drug types and present preclinical evidence that c-Jun inhibition is an attractive therapeutic target to prevent CIPN. View Publication