搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

How BMP signaling integrates into and destabilizes the pluripotency circuitry of human pluripotent stem cells (hPSCs) to initiate differentiation into individual germ layers is a long-standing puzzle. Here we report muscle segment homeobox 2 (MSX2),a homeobox transcription factor of msh family,as a direct target gene of BMP signaling and a master mediator of hPSCs' differentiation to mesendoderm. Enforced expression of MSX2 suffices to abolish pluripotency and induce directed mesendoderm differentiation of hPSCs,while MSX2 depletion impairs mesendoderm induction. MSX2 is a direct target gene of the BMP pathway in hPSCs,and can be synergistically activated by Wnt signals via LEF1 during mesendoderm induction. Furthermore,MSX2 destabilizes the pluripotency circuitry through direct binding to the SOX2 promoter and repression of SOX2 transcription,while MSX2 controls mesendoderm lineage commitment by simultaneous suppression of SOX2 and induction of NODAL expression through direct binding and activation of the Nodal promoter. Interestingly,SOX2 can promote the degradation of MSX2 protein,suggesting a mutual antagonism between the two lineage-specifying factors in the control of stem cell fate. Together,our findings reveal crucial new mechanisms of destabilizing pluripotency and directing lineage commitment in hPSCs. View Publication

We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD),a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91(phox) deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production,reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs,with subsequent selection and full characterization to ensure no off-target changes,holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease-mediated gene targeting of a single-copy gp91(phox) therapeutic minigene into one allele of the safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91(phox) and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs." View Publication

We employed Fourier transform infrared (FTIR) microspectroscopy to investigate the effects of different tissue culture environments on the FTIR spectra of undifferentiated human embryonic stem cells (hESCs) and their differentiated progeny. First we tested whether there were any possible spectral artifacts resulting from the use of transflectance measurements by comparing them with transmission measurements and found no evidence of these concluding that the lack of any differences resulted from the homogeneity of the dried cytospun cellular monolayers. We found that hESCs that were enzymatically passaged onto mouse embryonic fibroblasts (MEFs) in KOSR based hESC medium,hESCs enzymatically passaged onto Matrigel in mTESR medium and hESCs mechanically passaged onto MEFs in KOSR-based hESC medium,possessed unique FTIR spectroscopic signatures that reflect differences in their macromolecular chemistry. Further,these spectroscopic differences persisted even upon differentiation towards mesendodermal lineages. Our results suggest that FTIR microspectroscopy is a powerful,objective,measurement modality that complements existing methods for studying the phenotype of hESCs and their progeny,particularly changes induced by the cellular environment. View Publication

BackgroundMyeloid-derived suppressor cells (MDSCs) in tumor microenvironment reduce the efficacy of immunotherapy. PKN2 plays a role in colon cancer,but its function in esophageal cancer (EC) remains unclear. This study investigated PKN2 expression in MDSCs derived from EC tissues and determined whether PKN2 regulates immunosuppressive activity of MDSCs by mediating fatty acid oxidation (FAO).Materials and methodsPKN2 expression was determined in GEO database,EC patients,and 4-NQO-induced EC mice,as well as in different types of immune cells. The effect of PKN2 on the function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) was investigated by co-culture of PMN-MDSCs and CD4+/CD8+ T cells. The co-culture of patient-derived organoids and autologous immune cells was performed to observe the effect of PKN2 on the immunosuppressive function of PMN-MDSCs.ResultsPKN2 is highly expressed in EC tumor tissues compared to normal tissues,especially in tumor-infiltrated PMN-MDSCs. Overexpressing PKN2 in PMN-MDSCs contributes to the immunosuppressive activity of PMN-MDSCs in vitro. PKN2-overexpressing PMN-MDSCs inhibited the killing ability of cytotoxic T lymphocytes and promoted EC organoid growth. PKN2 promotes FAO in PMN-MDSCs via CPT1B (a key enzyme of FAO). Mechanistically,PKN2 promotes CPT1B transcription by upregulating STAT3 phosphorylation.ConclusionsPKN2 expression was increased in PMN-MDSCs derived from human and mouse EC tissues. PKN2 plays a role in enhancing the immunosuppressive activity of PMN-MDSCs by facilitating STAT3 phosphorylation and CPT1B transcription,which in turn leads to increased CPT1B-mediated FAO in PMN-MDSCs. Targeted inhibition of PKN2 is expected to improve immunotherapeutic efficacy in EC patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01132-6. View Publication

Defects in apoptosis contribute to poor outcome in pediatric acute lymphoblastic leukemia (ALL),calling for novel strategies that counter apoptosis resistance. Here,we demonstrate for the first time that small molecule inhibitors of the antiapoptotic protein XIAP cooperate with TRAIL to induce apoptosis in childhood acute leukemia cells. XIAP inhibitors at subtoxic concentrations,but not a structurally related control compound,synergize with TRAIL to trigger apoptosis and to inhibit clonogenic survival of acute leukemia cells,whereas they do not affect viability of normal peripheral blood lymphocytes,suggesting some tumor selectivity. Analysis of signaling pathways reveals that XIAP inhibitors enhance TRAIL-induced activation of caspases,loss of mitochondrial membrane potential,and cytochrome c release in a caspase-dependent manner,indicating that they promote a caspase-dependent feedback mitochondrial amplification loop. Of note,XIAP inhibitors even overcome Bcl-2-mediated resistance to TRAIL by enhancing Bcl-2 cleavage and Bak conformational change. Importantly,XIAP inhibitors kill leukemic blasts from children with ALL ex vivo and cooperate with TRAIL to induce apoptosis. In vivo,they significantly reduce leukemic burden in a mouse model of pediatric ALL engrafted in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Thus,XIAP inhibitors present a promising novel approach for apoptosis-based therapy of childhood ALL. View Publication

In the present study,we assessed the susceptibility of freshly isolated neuroblastoma cells to killing mediated by normal human natural killer (NK) cells and analyzed the receptor-ligand interactions that regulate this event. We show that killing of freshly isolated neuroblasts,similar to neuroblastoma cell lines,involves NKp46 and NKp30 (natural cytotoxicity receptors). However,freshly isolated neuroblasts were generally more resistant to NK-mediated lysis than conventional neuroblastoma cell lines. Moreover,a significant heterogeneity in susceptibility to lysis existed among neuroblastomas derived from different patients. Remarkably,susceptibility to lysis directly correlated with the surface expression,on neuroblasts,of poliovirus receptor [PVR (CD155)],a ligand for the DNAX accessory molecule-1 [DNAM-1 (CD226)] triggering receptor expressed by NK cells. Indeed,PVR-expressing neuroblastomas were efficiently killed by NK cells. Moreover,monoclonal antibody-mediated masking of either DNAM-1 (on NK cells) or PVR (on neuroblasts) resulted in strong inhibition of tumor cell lysis. Thus,assessment of the PVR surface levels may represent a novel useful criterion to predict the susceptibility/resistance of neuroblastomas to NK-mediated killing. View Publication

Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress,cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62),but its role in the regulation of autophagy is controversial. Here,we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK,thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly,p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV,and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy. View Publication

Enterochromaffin (EC) cells constitute the largest population of intestinal epithelial enteroendocrine (EE) cells. EC cells are proposed to be specialized mechanosensory cells that release serotonin in response to epithelial forces,and thereby regulate intestinal fluid secretion. However,it is unknown whether EE and EC cells are directly mechanosensitive,and if so,what the molecular mechanism of their mechanosensitivity is. Consequently,the role of EE and EC cells in gastrointestinal mechanobiology is unclear. Piezo2 mechanosensitive ion channels are important for some specialized epithelial mechanosensors,and they are expressed in mouse and human EC cells. Here,we use EC and EE cell lineage tracing in multiple mouse models to show that Piezo2 is expressed in a subset of murine EE and EC cells,and it is distributed near serotonin vesicles by superresolution microscopy. Mechanical stimulation of a subset of isolated EE cells leads to a rapid inward ionic current,which is diminished by Piezo2 knockdown and channel inhibitors. In these mechanosensitive EE cells force leads to Piezo2-dependent intracellular Ca2+ increase in isolated cells as well as in EE cells within intestinal organoids,and Piezo2-dependent mechanosensitive serotonin release in EC cells. Conditional knockout of intestinal epithelial Piezo2 results in a significant decrease in mechanically stimulated epithelial secretion. This study shows that a subset of primary EE and EC cells is mechanosensitive,uncovers Piezo2 as their primary mechanotransducer,defines the molecular mechanism of their mechanotransduction and mechanosensitive serotonin release,and establishes the role of epithelial Piezo2 mechanosensitive ion channels in regulation of intestinal physiology. View Publication

Rotavirus (RV),a primary cause of diarrhea-related mortality in 2021,has been shown to damage intestinal epithelial cells while upregulating intestinal stem cells (ISCs) activities. ISCs within the crypt niche drive the continuous self-renewal of intestinal epithelium,preserving its barrier functions. Paneth cells secrete antimicrobial peptide and signaling molecules within the intestine crypt,thereby playing a crucial role in intestinal immune defense and providing ISCs functional support. However,the regulatory function of Paneth cells under pathological conditions,such as RV infection,remains unclear. To determine the impact of RV infection on Paneth cells and how Paneth cells regulate ISCs during intestinal injury repair. We constructed a reference genome for the RV enteric cytopathogenic human orphan virus strain and reanalyzed published single-cell RNA sequencing data to investigate Paneth cell responses to RV-induced intestinal injury. We derived Paneth-ISC communication networks using CellChat,tracked ISC differentiation with pseudotime analysis,and validated our findings in leucine-rich repeat-containing G protein-coupled receptor 5-enhanced green fluorescent protein-internal ribosomal entry site-Cre recombinase estrogen receptor variant 2 mice and organoids via immunofluorescence,flow cytometry,and reverse transcription quantitative polymerase chain reaction. We found that RV directly infects Paneth cells,leading to a reduction in mature Paneth cells and an increase in kallikrein 1-high immature Paneth cells. Paneth-ISC communication was significantly enhanced. In particular,the bone morphogenic protein 7 (BMP7)-activin A receptor type 2B/BMP receptor type 1A-Smad pathway was upregulated post-infection,suggesting that Paneth cells suppress excessive ISC proliferation. Functional validation confirmed activation of this pathway. Paneth cells regulate ISC proliferation during RV infection by activating BMP7 signaling,limiting excessive stem cell expansion and preserving crypt homeostasis for effective epithelial repair. View Publication

AbstractBack to TopbackslashnNeurons produced from stem cells have emerged as a tool to identify new therapeutic targets for neurological diseases such as amyotrophic lateral sclerosis (ALS). However,it remains unclear to what extent these new mechanistic insights will translate to animal models,an important step in the validation of new targets. Previously,we found that glia from mice carrying the SOD1G93A mutation,a model of ALS,were toxic to stem cell–derived human motor neurons. We use pharmacological and genetic approaches to demonstrate that the prostanoid receptor DP1 mediates this glial toxicity. Furthermore,we validate the importance of this mechanism for neural degeneration in vivo. Genetic ablation of DP1 in SOD1G93A mice extended life span,decreased microglial activation,and reduced motor neuron loss. Our findings suggest that blocking DP1 may be a therapeutic strategy in ALS and demonstrate that discoveries from stem cell models of disease can be corroborated in vivo. View Publication

Achieving a cure is an urgent need for patients with advanced solid tumors. Here,we discover that oncolytic virus (OV) infection enhances IL-18 receptor expression but fails to increase IL-18 ligand expression. Therefore,we engineer armed oncolytic alphavirus M1 expressing wild-type IL-18 (wtIL-18) or a mutant variant (mutIL-18) that evades IL-18 binding protein (IL-18BP) while maintaining IL-18 receptor (IL-18R) binding. Intravenous administration of M1-mutIL-18 suppresses the growth of multiple advanced solid tumors in C57BL/6 and BALB/c mouse models and promotes long-term systemic immune memory. Mechanistically,armed M1-mutIL-18 enhances directed clonal expansion and differentiation of CD8+ T cells and sustains IFN-γ production. Thus,armed M1-mutIL-18 promotes dendritic cell (DC) activation,priming and activation of CD8+ T cells in lymphatic organs,and infiltration of IL-18R+ CD8+ T cells in the tumor microenvironment,establishing a positive feedback loop. We further show that a PD-L1 inhibitor enhances the anti-tumor efficacy of mutIL-18 OVs. These results highlight the importance of the IL-18 pathway in oncolytic virus therapy and implicate reprogramming ligand-receptor interaction as an effective strategy for immunotherapy. Immunotherapy holds great potential,although strategies for durable responses against solid tumors are still needed. Here,the authors combine oncolytic virus (OV) engineering and reprogramming of the IL-18 pathway,showing that armed OVs expressing a decoy-resistant IL-18 elicit anti-tumor immunity and long-term immunological memory against multiple refractory tumors in mice. View Publication

The cellular reservoir for latent human cytomegalovirus (HCMV) in the hematopoietic compartment,and the mechanisms governing a latent infection and reactivation from latency are unknown. Previous work has demonstrated that HCMV infects CD34+ progenitors and expresses a limited subset of viral genes. The outcome of HCMV infection may depend on the cell subpopulations infected within the heterogeneous CD34+ compartment. We compared HCMV infection in well-defined CD34+ cell subpopulations. HCMV infection inhibited hematopoietic colony formation from CD34+/CD38- but not CD34+/c-kit+ cells. CD34+/CD38- cells transiently expressed a large subset of HCMV genes that were not expressed in CD34+/c-kit+ cells or cells expressing more mature cell surface phenotypes. Although viral genomes were present in infected cells,viral gene expression was undetectable by 10 days after infection. Importantly,viral replication could be reactivated by coculture with permissive fibroblasts only from the CD34+/CD38- population. Strikingly,a subpopulation of CD34+/CD38- cells expressing a stem cell phenotype (lineage-/Thy-1+) supported a productive HCMV infection. These studies demonstrate that the outcome of HCMV infection in the hematopoietic compartment is dependent on the nature of the cell subpopulations infected and that CD34+/CD38- cells support an HCMV infection with the hallmarks of latency. View Publication