技术资料

There is no consensus in the stem cell field as to what constitutes the mature cardiac myocyte. Thus,helping formalize a molecular signature for cardiac myocyte maturation would advance the field. In the mammalian heart,inactivation of the fetal" TNNI gene� View Publication

The ability to derive retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) has led to numerous advances in the field of retinal research,with great potential for the use of hPSC-derived RGCs for studies of human retinal development,in vitro disease modeling,drug discovery,as well as their potential use for cell replacement therapeutics. Of all these possibilities,the use of hPSC-derived RGCs as a human-relevant platform for in vitro disease modeling has received the greatest attention,due to the translational relevance as well as the immediacy with which results may be obtained compared to more complex applications like cell replacement. While several studies to date have focused upon the use of hPSC-derived RGCs with genetic variants associated with glaucoma or other optic neuropathies,many of these have largely described cellular phenotypes with only limited advancement into exploring dysfunctional cellular pathways as a consequence of the disease-associated gene variants. Thus,to further advance this field of research,in the current study we leveraged an isogenic hPSC model with a glaucoma-associated mutation in the Optineurin (OPTN) protein,which plays a prominent role in autophagy. We identified an impairment of autophagic-lysosomal degradation and decreased mTORC1 signaling via activation of the stress sensor AMPK,along with subsequent neurodegeneration in OPTN(E50K) RGCs differentiated from hPSCs,and have further validated some of these findings in a mouse model of ocular hypertension. Pharmacological inhibition of mTORC1 in hPSC-derived RGCs recapitulated disease-related neurodegenerative phenotypes in otherwise healthy RGCs,while the mTOR-independent induction of autophagy reduced protein accumulation and restored neurite outgrowth in diseased OPTN(E50K) RGCs. Taken together,these results highlighted that autophagy disruption resulted in increased autophagic demand which was associated with downregulated signaling through mTORC1,contributing to the degeneration of RGCs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-024-01872-2. View Publication

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs exhibit basal-apical polarity,junctional complexes,integrin-dependent matrix adhesion,and E-cadherin-dependent cell-cell adhesion,all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures,programmed cell death is commonly observed. If individualized human ESCs are prevented from reattaching and forming colonies,their viability is significantly reduced. Here,we show that actin-myosin contraction is a critical effector of the cell death response to human ESC dissociation. Inhibition of myosin heavy chain ATPase,downregulation of myosin heavy chain,and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ESCs. ROCK inhibition decreases phosphorylation of myosin light chain,suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ESCs. View Publication

In many mammalian cells brefeldin A interferes with mechanisms that keep the Golgi appartus separate from the endoplasmic reticulum. The earliest effect of brefeldin A is release of the coat protein beta-COP from the Golgi. This release is blocked by pretreatment with GTP-gamma S or AlF4- (ref. 12). The AlF4- ion activates heterotrimeric G proteins but not proteins of the ras superfamily,suggesting that a heterotrimeric G protein might control membrane transfer from the endoplasmic reticulum to the Golgi. We report here that mastoparan,a peptide that activates heterotrimeric G proteins,promotes binding of beta-COP to Golgi membranes in vitro and antagonizes the effect of brefeldin A on beta-COP in perforated cells and on isolated Golgi membranes. This inhibition is greatly diminished if cells are pretreated with pertussis toxin before perforation. Thus,a heterotrimeric G protein of the Gi/Go subfamily regulates association of coat components with Golgi membranes. View Publication

U937 monocytic cells are shown here to express a range of PDE4,cAMP-specific phosphodiesterase (PDE) isoenzymes: the long isoenzymes,PDE4A4,PDE4D5 and PDE4D3,plus the short isoenzyme,PDE4B2. These isoenzymes provide around 76% of the total cAMP PDE activity of U937 cells. The specific activities of the total PDE4A,PDE4B and PDE4D activities were 0.63+/-0.09,8.8+/-0.2 and 34.4+/-2.9 pmol/min per mg of protein respectively. The PDE4 selective inhibitor,rolipram,inhibited immunopurified PDE4B and PDE4D activities similarly,with IC(50) values of approx. 130 nM and 240 nM respectively. In contrast,rolipram inhibited immunopurified PDE4A activity with a dramatically lower IC(50) value of around 3 nM. Rolipram increased phosphorylation of cAMP-response-element-binding protein (CREB) in U937 cells in a dose-dependent fashion,which implied the presence of both high affinity (IC(50) value approx. 1 nM) and low affinity (IC(50) value approx. 120 nM) components. Rolipram dose-dependently inhibited the interferon-gamma (IFN-gamma)-stimulated phosphorylation of p38 mitogen-activated protein (MAP) kinase in a simple monotonic fashion with an IC(50) value of approx. 290 nM. On this basis,it is suggested that rolipram inhibition of PDE4A4 is involved in regulating CREB phosphorylation but not IFN-gamma-stimulated p38 MAP kinase phosphorylation. PDE4A4 was also selectively activated by challenge of U937 cells with either bacterial lipopolysaccharide (LPS) or IFN-gamma through a process which was attenuated by both wortmannin and rapamycin. It is proposed that the PDE4A4 isoform is involved in compartmentalized cAMP signalling responses in U937 monocytes. View Publication

BACKGROUND Myeloid-derived suppressor cells (MDSCs) can potently inhibit T-cell activity,promote growth and metastasis of tumor and contribute to resistance to immunotherapy. Targeting MDSCs to alleviate their protumor functions and immunosuppressive activities is intimately associated with cancer immunotherapy. Natural killer (NK) cells can engage in crosstalk with multiple myeloid cells to alter adaptive immune responses,triggering T-cell immunity. However,whether the NK-cell-MDSC interaction can modulate the T-cell immune response requires further study. Cryo-thermal therapy could induce the maturation of MDSCs by creating an acute inflammatory environment to elicit a CD4+ Th1-dominant immune response,but the mechanism regulating this process remains unclear. METHODS NK cells were depleted and NKG2D was blocked with monoclonal antibodies in vivo. MDSCs,NK cells and T cells were assessed by flow cytometry and isolated by magnetic-activated cell sorting (MACS). MDSCs and NK cells were cocultured with T cells to determine their immunological function. The transcriptional profiles of MDSCs were measured by qRT-PCR and RNA-sequencing. Isolated NK cells and MDSCs by MACS were cocultured to study the viability and maturation of MDSCs regulated by NK cells. TIMER was used to comprehensively examine the immunological,clinical,and genomic features of tumors. RESULTS NK-cell activation after cryo-thermal therapy decreased MDSC accumulation and reprogrammed immunosuppressive MDSCs toward a mature phenotype to promote T cell antitumor immunity. Furthermore,we discovered that NK cells could kill MDSCs via the NKG2D-NKG2DL axis and promote MDSC maturation by interferon gamma (IFN-$\gamma$) in response to NKG2D. In addition,CD4+ Th1-dominant antitumor immune response was dependent on NKG2D,which promoted the major histocompatibility complex …¡ pathway of MDSCs. High activated NK-cell infiltration and NKG2D level in tumors were positively correlated with better clinical outcomes. CONCLUSIONS Cryo-thermal therapy induces effective CD4+ Th1-dominant antitumor immunity by activating NK cells to reprogram MDSCs,providing a promising therapeutic strategy for cancer immunotherapy. View Publication

The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens,but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells,we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs,40{\%} of antigen-specific SED B cells bind antigen within 2 h,whereas unspecifc cells do not,indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice,but is unperturbed in mice depleted of classical dendritic cells (DC). Thus,we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses,and should be taken into account when developing mucosal vaccines. View Publication

Accumulating evidence suggests that the regulation of gene expression by histone lysine methylation is crucial for several biological processes. The histone lysine methyltransferase G9a is responsible for the majority of dimethylation of histone H3 at lysine 9 (H3K9me2) and is required for the efficient repression of developmentally regulated genes during embryonic stem cell differentiation. However,whether G9a plays a similar role in adult cells is still unclear. We identify a critical role for G9a in CD4(+) T helper (Th) cell differentiation and function. G9a-deficient Th cells are specifically impaired in their induction of Th2 lineage-specific cytokines IL-4,IL-5,and IL-13 and fail to protect against infection with the intestinal helminth Trichuris muris. Furthermore,G9a-deficient Th cells are characterised by the increased expression of IL-17A,which is associated with a loss of H3K9me2 at the Il17a locus. Collectively,our results establish unpredicted and complex roles for G9a in regulating gene expression during lineage commitment in adult CD4(+) T cells. View Publication

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors,and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions,yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore,we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1,and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors,leading to target cell death. View Publication

UNLABELLED Parkinson's disease (PD) is characterized by the accumulation of α-synuclein (α-syn) within Lewy body inclusions in the nervous system. There are currently no disease-modifying therapies capable of reducing α-syn inclusions in PD. Recent data has indicated that loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase) represent an important risk factor for PD,and can lead to α-syn accumulation. Here we use a small-molecule modulator of GCase to determine whether GCase activation within lysosomes can reduce α-syn levels and ameliorate downstream toxicity. Using induced pluripotent stem cell (iPSC)-derived human midbrain dopamine (DA) neurons from synucleinopathy patients with different PD-linked mutations,we find that a non-inhibitory small molecule modulator of GCase specifically enhanced activity within lysosomal compartments. This resulted in reduction of GCase substrates and clearance of pathological α-syn,regardless of the disease causing mutations. Importantly,the reduction of α-syn was sufficient to reverse downstream cellular pathologies induced by α-syn,including perturbations in hydrolase maturation and lysosomal dysfunction. These results indicate that enhancement of a single lysosomal hydrolase,GCase,can effectively reduce α-syn and provide therapeutic benefit in human midbrain neurons. This suggests that GCase activators may prove beneficial as treatments for PD and related synucleinopathies. SIGNIFICANCE STATEMENT The presence of Lewy body inclusions comprised of fibrillar α-syn within affected regions of PD brain has been firmly documented,however no treatments exist that are capable of clearing Lewy bodies. Here,we used a mechanistic-based approach to examine the effect of GCase activation on α-syn clearance in human midbrain DA models that naturally accumulate α-syn through genetic mutations. Small molecule-mediated activation of GCase was effective at reducing α-syn inclusions in neurons,as well as associated downstream toxicity,demonstrating a therapeutic effect. Our work provides an example of how human iPSC-derived midbrain models could be used for testing potential treatments for neurodegenerative disorders,and identifies GCase as a critical therapeutic convergence point for a wide range of synucleinopathies. View Publication

The ability of somatic cells to reprogram their ATP-generating machinery into a Warburg-like glycolytic metabotype while overexpressing stemness genes facilitates their conversion into either induced pluripotent stem cells (iPSCs) or tumor-propagating cells. AMP-activated protein kinase (AMPK) is a metabolic master switch that senses and decodes intracellular changes in energy status; thus,we have evaluated the impact of AMPK activation in regulating the generation of iPSCs from nonstem cells of somatic origin. The indirect and direct activation of AMPK with the antidiabetic biguanide metformin and the thienopyridone A-769662,respectively,impeded the reprogramming of mouse embryonic and human diploid fibroblasts into iPSCs. The AMPK activators established a metabolic barrier to reprogramming that could not be bypassed,even through p53 deficiency,a fundamental mechanism to greatly improve the efficiency of stem-cell production. Treatment with metformin or A-769662 before the generation of iPSC colonies was sufficient to drastically decrease iPSC generation,suggesting that AMPK activation impedes early stem cell genetic reprogramming. Monitoring the transcriptional activation status of each individual reprogramming factor (i.e.,Oct4,Sox2,Klf4 and c-Myc) revealed that AMPK activation notably prevented the transcriptional activation of Oct4,the master regulator of the pluripotent state. AMPK activation appears to impose a normalized metabolic flow away from the required pro-immortalizing glycolysis that fuels the induction of stemness and pluripotency,endowing somatic cells with an energetic infrastructure that is protected against reprogramming. AMPK-activating anti-reprogramming strategies may provide a roadmap for the generation of novel cancer therapies that metabolically target tumor-propagating cells. View Publication

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab,the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance. View Publication