技术资料

Osteoporosis diagnoses are increasing in the ageing population,and although some treatments exist,these have several disadvantages,highlighting the need to identify new drug targets. G protein-coupled receptors (GPCRs) are transmembrane proteins whose surface expression and extracellular activation make them desirable drug targets. Our previous studies have identified 144 GPCR genes to be expressed in primary human osteoclasts,which could provide novel drug targets. The development of high-throughput assays to assess osteoclast activity would improve the efficiency at which we could assess the effect of GPCR activation on human bone cells and could be utilised for future compound screening. Here,we assessed the utility of a high-content imaging (HCI) assay that measured cytoplasmic-to-nuclear translocation of the nuclear factor of activated T cells-1 (NFATc1),a transcription factor that is essential for osteoclast differentiation,and resorptive activity. We first demonstrated that the HCI assay detected changes in NFATc1 nuclear translocation in human primary osteoclasts using GIPR as a positive control,and then developed an automated analysis platform to assess NFATc1 in nuclei in an efficient and unbiased manner. We assessed six GPCRs simultaneously and identified four receptors (FFAR2,FFAR4,FPR1 and GPR35) that reduced osteoclast activity. Bone resorption assays and measurements of TRAP activity verified that activation of these GPCRs reduced osteoclast activity,and that receptor-specific antagonists prevented these effects. These studies demonstrate that HCI of NFATc1 can accurately assess osteoclast activity in human cells,reducing observer bias and increasing efficiency of target detection for future osteoclast-targeted osteoporosis therapies. View Publication

Myelodysplastic syndrome (MDS) is a malignant hematologic disorder with limited curative options,primarily reliant on hematopoietic stem cell transplantation. Anemia,a prevalent symptom of MDS,has few effective treatment strategies. Realgar,though known for its therapeutic effects on MDS,remains poorly understood in terms of its mechanism of action. In this study,both in vivo and in vitro experiments were conducted using Realgar and its primary active component,As 2 S 2,to examine their impact on mouse erythroblasts at the single-cell level. Realgar treatment significantly altered the transcriptional profiles and cellular composition of bone marrow in mice,both in vivo and in vitro. Differentially expressed genes in erythroblasts regulated by Realgar were identified,unveiling potential regulatory functions and signaling pathways,such as heme biosynthesis,hemoglobin production,oxygen binding,IL-17 signaling,and MAPK pathways. These findings suggest that Realgar enhances the differentiation of erythroblasts in mouse bone marrow and improves overall blood cell counts. This work offers preliminary insights into Realgar’s mechanisms,expands the understanding of this mineral medicine,and may inform strategies to optimize its therapeutic potential in hematologic diseases. The online version contains supplementary material available at 10.1186/s12935-025-03768-0. View Publication

Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. We studied the in vitro response of neural cultures exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex,cerebellum and remaining areas. First,each brain region was evaluated with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second,the cerebral regions were subjected to the decellularization process and their respective characterization using molecular,histological,and ultrastructural techniques. Third,the levels of neurotrophic factors in the decellularized brain scaffold were analyzed. Fourth,we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells,as a unidirectional model of differentiation. Finally,in vitro studies were carried out to evaluate the cell recovery capacity of brain decellularized ECM under stroke-mimetic conditions. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and,in addition,it possesses a combination of structural and biochemical signals (e.g.,neurotrophic factors) that are capable of inducing cell phenotypic changes and promote viability and cell recovery in a stroke/ischemia model in vitro. The online version contains supplementary material available at 10.1038/s41598-025-95656-w. View Publication

Genetically encoded calcium ion (Ca 2+ ) indicators (GECIs) are widely-used molecular tools for functional imaging of Ca 2+ dynamics and neuronal activities with single-cell resolution. Here we report the design and development of two far-red fluorescent GECIs,FR-GECO1a and FR-GECO1c,based on the monomeric far-red fluorescent proteins mKelly1 and mKelly2. FR-GECOs have excitation and emission maxima at ~596 nm and ~644 nm,respectively,display large responses to Ca 2+ in vitro (Δ F / F 0 = 6 for FR-GECO1a,18 for FR-GECO1c),are bright under both one-photon and two-photon illumination,and have high affinities (apparent K d = 29 nM for FR-GECO1a,83 nM for FR-GECO1c) for Ca 2+ . FR-GECOs offer sensitive and fast detection of single action potentials in neurons,and enable in vivo all-optical manipulation and measurement of cellular activities in combination with optogenetic actuators. Subject terms: Fluorescent proteins,Optogenetics,Zebrafish,Molecular neuroscience,Calcium signalling View Publication

Studying ciliary genes in the context of the human central nervous system is crucial for understanding the underlying causes of neurodevelopmental ciliopathies. Here,we use pluripotent stem cell-derived spinal organoids to reveal distinct functions of the ciliopathy gene RPGRIP1L in humans and mice,and uncover an unexplored role for cilia in human axial patterning. Previous research has emphasized Rpgrip1l critical functions in mouse brain and spinal cord development through the regulation of SHH/GLI pathway. Here,we show that RPGRIP1L is not required for SHH activation or motoneuron lineage commitment in human spinal progenitors and that this feature is shared by another ciliopathy gene,TMEM67 . Furthermore,human RPGRIP1L -mutant motoneurons adopt hindbrain and cervical identities instead of caudal brachial identity. Temporal transcriptome analysis reveals that this antero-posterior patterning defect originates in early axial progenitors and correlates with cilia loss. These findings provide important insights into the role of cilia in human neural development. Subject terms: Ciliogenesis,Pattern formation,Pluripotent stem cells,Neurogenesis View Publication

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. We previously discovered that heme oxygenase 1 (HO1) is crucial for chemoresistance in AML,but the detailed molecular mechanism of that remains unclear. RNA sequencing was conducted to assess transcriptomic changes in three pairs of AML cells after regulating the expression of HO1. The molecular mechanism by which HO1 induces gilteritinib resistance in FLT3-ITD (FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD)) AML was evaluated by quantitative real-time PCR (qRT-PCR),CCK-8,flow cytometry,and western blotting. FLT3-ITD AML mouse models were established to investigate the effects of HO1 expression on gilteritinib resistance in vivo. In these three pairs of AML cells,we discovered that HO1-mediated drug resistance is connected to the interleukin-4-mediated signaling pathway (specifically STAT6) only in MV4-11 cells with the FLT3-ITD mutation. Further findings revealed that HO1 overexpression confers gilteritinib resistance in FLT3-ITD AML cell lines and primary individual specimens. While suppression of HO1 sensitized FLT3-ITD AML cell lines and primary individual specimens to gilteritinib. Mechanistically,western blotting and flow cytometry confirmed that HO1-mediated gilteritinib resistance is related to STAT6 phosphorylation in FLT3-ITD AML cell lines and primary individual specimens. Moreover,tumor-bearing mice were employed to determine that HO1 overexpression conferred gilteritinib resistance in vivo. Collectively,these studies illustrate that HO1 may act as a successful treatment target for gilteritinib-resistant FLT3-ITD AML patients. The online version contains supplementary material available at 10.1186/s12935-025-03757-3. View Publication

Trogocytosis is the process by which a recipient cell siphons small membrane fragments and proteins from a donor cell and can be utilized by cancer cells to avoid immune detection. We observed lymphocyte specific protein expressed by triple negative breast cancer (TNBC) cells via immunofluorescence imaging of patient samples. Image analysis of Cluster of Differentiation 45RA (CD45RA) expression,a naïve T cell specific protein,revealed that all stages of TNBCs express CD45RA. Flow cytometry revealed TNBC cells trogocytose CD45 protein from T cells. We also showed that the acquisition of these lymphoid markers is contact dependent. Confocal and super-resolution imaging further revealed CD45+ spherical structures containing T cell genomic DNA inside TNBC cells after co-culture. Trogocytosis between T cells and TNBC cells altered tumor cell expression of PTPRC,the gene that encodes for CD45. Our results revealed that CD45 is obtained by TNBC cells from T cells via trogocytosis and that TNBC cells express CD45 intracellularly and on the membrane. View Publication

Designer receptors exclusively activated by designer drugs (DREADDs) are established tools for modulating neuronal activity. Calcium-mobilizing DREADD hM3Dq has been widely used to enhance neuronal activity. hM3Dq activates the Gq protein signaling cascade and mimics the action of native Gq protein-coupled receptors such as muscarinic m1 and m3 receptors leading to calcium release from intracellular storages. Depolarization evoked by increased intracellular calcium levels is an important factor for neuronal maturation. Here,we used repetitive activation of biolistically overexpressed hM3Dq to increase the activity of individual neurons differentiating in organotypic slice cultures of rat visual cortex. HM3Dq was activated by 3 μM clozapine-N-oxide (CNO) dissolved in H 2 O. Transfectants expressing hM3Dq mock-stimulated with H 2 O served as batch-internal controls. Pyramidal cells and multipolar interneurons were analyzed after treatment from DIV 5–10,DIV 10–20,and DIV 15–20 to investigate if Gq signaling is involved in dendritic maturation. Results show that hM3Dq activation accelerated the maturation of apical dendrites of L2/3 pyramidal cells in the early,but no longer in the later time windows. In contrast,dendritic dimensions of L5/6 pyramidal cells and interneurons were not altered at DIV 10. These findings suggest a growth-promoting role of activated Gq signaling selectively for early postnatal L2/3 pyramidal cells. Unexpectedly,hM3Dq activation from DIV 10–20 reduced the dendritic complexity of L5/6 pyramidal cells and multipolar interneurons. Together,results suggest a role of Gq signaling for neuronal differentiation and support evidence that it may also limit dendritic growth. View Publication

To produce pluripotent stem cells from peripheral blood mononuclear cells (PBMCs) of a patient with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and culture and differentiate them into vascular organoids,producing a disease model for cerebral small vessel disease (CSVD). (1) PMBCs from patients clinically diagnosed with CADASIL ( NOTCH3 p.R141C) were induced to differentiate into pluripotent stem cells (iPSCs); the quality and differentiation ability of the iPSCs were determined. (2) CADASIL-derived iPSCs and control iPSCs were cultured and differentiated into vascular organoids. The differences in the morphological structure of the two differentiated groups of vascular organoids were observed,and both were identified. (1) No mycoplasma infections were detected in the iPSCs prepared from the PBMCs of patients with CADASIL. The short tandem repeat (STR) identification verified that the iPSCs originated from the patient,and the karyotype was normal. Flow cytometry and immunofluorescence detection revealed that the iPSCs expressed SSEA4,OCT4,and NANOG stem proteins. Tri-germ differentiation testing confirmed that the iPSCs expressed the endoderm markers SOX17 and FOXA2,the mesoderm markers Brachyury and α-SMA,and the ectoderm markers Pax6 and β-III Tubulin. (2) CADASIL-derived iPSCs and control iPSCs were induced to differentiate and produce endothelial networks and vascular networks,ultimately forming vascular organoids. Compared with control vascular organoids,CADASIL vascular organoids exhibited lower growth density,earlier blood vessel sprouting,longer and thinner vascular filaments,and smaller final vascular organoids. The vascular organoids from the two sources expressed the endothelial cell marker CD31,the vascular smooth muscle marker α-SMA,and the pericyte marker PDGFR-β. Reprogramming technology can be used to induce PBMCs to become iPSCs,and a CSVD disease model can be successfully constructed by culturing and differentiating the iPSCs into CADASIL vascular organoids. The NOTCH3 p.R141C mutation suppresses the vascular differentiation process in CADASIL. View Publication

The search for an effective cell replacement therapy for diabetes has driven the development of “perfect” pancreatic islets from human pluripotent stem cells (hPSCs). These hPSC-derived pancreatic islet-like β cells can overcome the limitations for disease modelling,drug development and transplantation therapies in diabetes. Nevertheless,challenges remain in generating fully functional and mature β cells from hPSCs. This review underscores the significant efforts made by researchers to optimize various differentiation protocols aimed at enhancing the efficiency and quality of hPSC-derived pancreatic islets and proposes methods for their improvement. By emulating the natural developmental processes of pancreatic embryogenesis,specific growth factors,signaling molecules and culture conditions are employed to guide hPSCs towards the formation of mature β cells capable of secreting insulin in response to glucose. However,the efficiency of these protocols varies greatly among different human embryonic stem cell (hESC) and induced pluripotent stem cell (hiPSC) lines. This variability poses a particular challenge for generating patient-specific β cells. Despite recent advancements,the ultimate goal remains to develop a highly efficient directed differentiation protocol that is applicable across all genetic backgrounds of hPSCs. Although progress has been made,further research is required to optimize the protocols and characterization methods that could ensure the safety and efficacy of hPSC-derived pancreatic islets before they can be utilized in clinical settings. View Publication