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

Background: Human pluripotent stem cells (hPSCs),including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs),can undergo erythroid differentiation,offering a potentially invaluable resource for generating large quantities of erythroid cells. However,the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors,with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms. Methods: In this study,we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies,including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs,morphological analysis,quantitative real-time PCR (qRT-PCR),western blotting,flow cytometry,karyotyping,cytospin,Benzidine-Giemsa staining,immunofluorescence assay,and high-speed cell imaging analysis,were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation,as well as their differentiated erythroid cells. Results: Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells,whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin. Conclusions: VIM disappear during the normal maturation of erythroid cells,whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform,we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization. View Publication

Cell signaling plays a critical role in neurodevelopment,regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing,scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq,an integrated approach that aims to quantify cytoplasmic and nuclear proteins,including those with post-translational modifications,and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom neuro-focused antibody panels for simultaneous protein and scATAC-seq profiling on whole cells,alongside both experimental and computational strategies to incorporate transcriptomic measurements. We apply our workflow to cell lines,induced pluripotent stem cells,and months-old retinal and brain organoids to demonstrate its broad applicability. We show that Phospho-seq can provide insights into cellular states and trajectories,shed light on gene regulatory relationships,and help explore the causes and effects of diverse cell signaling in neurodevelopment. Here,the authors demonstrate Phospho-seq,a single-cell multiomics method capable of quantifying chromatin accessibility alongside intracellular proteins,including post-translationally modified proteins. Then,they apply Phospho-seq to organoid models of neurodevelopment. View Publication

The generation of iPSC-derived hepatocyte-like cells (HLCs) is a powerful tool for studying liver diseases,their therapy as well as drug development. iPSC-derived disease models benefit from their diverse origin of patients,enabling the study of disease-associated mutations and,when considering more than one iPSC line to reflect a more diverse genetic background compared to immortalized cell lines. Unfortunately,the use of iPSC-derived HLCs is limited due to their lack of maturity and a rather fetal phenotype. Commercial kits and complicated 3D-protocols are cost- and time-intensive and hardly useable for smaller working groups. In this study,we optimized our previously published protocol by fine-tuning the initial cell number,exchanging antibiotics and basal medium composition and introducing the small molecule forskolin during the HLC maturation step. We thereby contribute to the liver research field by providing a simple,cost- and time-effective 2D differentiation protocol. We generate functional HLCs with significantly increased HLC hallmark gene (ALB,HNF4?,and CYP3A4) and protein (ALB) expression,as well as significantly elevated inducible CYP3A4 activity. Graphical Abstract View Publication

BackgroundDendritic cell (DC)-mediated antigen presentation is essential for the priming and activation of tumor-specific T cells. However,few drugs that specifically manipulate DC functions are available. The identification of drugs targeting DC holds great promise for cancer immunotherapy.MethodsWe observed that type 1 conventional DCs (cDC1s) initiated a distinct transcriptional program during antigen presentation. We used a network-based approach to screen for cDC1-targeting therapeutics. The antitumor potency and underlying mechanisms of the candidate drug were investigated in vitro and in vivo.ResultsSitagliptin,an oral gliptin widely used for type 2 diabetes,was identified as a drug that targets DCs. In mouse models,sitagliptin inhibited tumor growth by enhancing cDC1-mediated antigen presentation,leading to better T-cell activation. Mechanistically,inhibition of dipeptidyl peptidase 4 (DPP4) by sitagliptin prevented the truncation and degradation of chemokines/cytokines that are important for DC activation. Sitagliptin enhanced cancer immunotherapy by facilitating the priming of antigen-specific T cells by DCs. In humans,the use of sitagliptin correlated with a lower risk of tumor recurrence in patients with colorectal cancer undergoing curative surgery.ConclusionsOur findings indicate that sitagliptin-mediated DPP4 inhibition promotes antitumor immune response by augmenting cDC1 functions. These data suggest that sitagliptin can be repurposed as an antitumor drug targeting DC,which provides a potential strategy for cancer immunotherapy. View Publication

Chimeric antigen receptor (CAR) T cells have shown remarkable response rates in hematological malignancies. In contrast,CAR T cell treatment of solid tumors is associated with several challenges,in particular the expression of most tumor-associated antigens at lower levels in vital organs,resulting in on-target/off-tumor toxicities. Thus,innovative approaches to improve the tumor specificity of CAR T cells are urgently needed. Based on the observation that many human solid tumors activate epidermal growth factor receptor (EGFR) on their surface through secretion of EGFR ligands,we developed an engineering strategy for CAR-binding domains specifically directed against the ligand-activated conformation of EGFR. We show,in several experimental systems,that the generated binding domains indeed enable CAR T cells to distinguish between active and inactive EGFR. We anticipate that this engineering concept will be an important step forward to improve the tumor specificity of CAR T cells directed against EGFR-positive solid cancers. View Publication

BACKGROUND Friedreich's ataxia (FRDA),a recessive neurodegenerative disorder commonly associated with hypertrophic cardiomyopathy,is caused by silencing of the frataxin (FXN) gene encoding the mitochondrial protein involved in iron-sulfur cluster biosynthesis. METHODS Application of our previously established FRDA human induced pluripotent stem cell (hiPSC) derived cardiomyocytes model as a platform to assess the efficacy of treatment with either the antioxidant coenzyme Q10 analog,idebenone (IDE) or the iron chelator,deferiprone (DFP),which are both under clinical trial. RESULTS DFP was able to more significantly suppress synthesis of reactive oxygen species (ROS) than IDE at the dosages of 25 $\$ and 10nM respectively which agreed with the reduced rate of intracellular accumulation of iron by DFP treatment from 25 to 50 $\$ With regard to cardiac electrical-contraction (EC) coupling function,decay velocity of calcium handling kinetics in FRDA-hiPSC-cardiomyocytes was significantly improved by DFP treatment but not by IDE. Further mechanistic studies revealed that DFP also modulated iron induced mitochondrial stress as reflected by mitochondria network disorganization and decline level of respiratory chain protein,succinate dehydrogenase (CxII) and cytochrome c oxidase (COXIV). In addition,iron-response protein (IRP-1) regulatory loop was overridden by DFP as reflected by resumed level of ferritin (FTH) back to basal level and the attenuated transferrin receptor (TSFR) mRNA level suppression thereby reducing further iron uptake. CONCLUSIONS DFP modulated iron homeostasis in FRDA-hiPSC-cardiomyocytes and effectively relieved stress-stimulation related to cardiomyopathy. The resuming of redox condition led to the significantly improved cardiac prime events,cardiac electrical-coupling during contraction. View Publication

Regenerative medicine is in need of solid,large animal models as a link between rodents and humans to evaluate the functionality,immunogenicity,and clinical safety of stem cell-derived cell types. The common marmoset (Callithrix jacchus) is an excellent large animal model,genetically close to humans and readily used worldwide in clinical research. Until now,only two groups showed the generation of induced pluripotent stem cells (iPSCs) from the common marmoset using integrating retroviral vectors. Therefore,we reprogrammed bone marrow-derived mesenchymal cells (MSCs) of adult marmosets in the presence of TAV,SB431542,PD0325901,and ascorbic acid via a novel,excisable lentiviral spleen focus-forming virus (SFFV)-driven quad-cistronic vector system (OCT3/4,KLF4,SOX2,C-MYC). Endogenous pluripotency markers like OCT3/4,KLF4,SOX2,C-MYC,LIN28,NANOG,and strong alkaline phosphatase signals were detected. Exogenous genes were silenced and additionally the cassette was removed with a retroviral Gag precursor system. The cell line could be cultured in absence of leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) and could be successfully differentiated into embryoid bodies and teratomas with presence of all three germ layers. Directed differentiation generated neural progenitors,megakaryocytes,adipocytes,chondrocytes,and osteogenic cells. Thus,all criteria for fully reprogrammed bone marrow-MSCs of a nonhuman primate with a genetically sophisticated construct could be demonstrated. These cells will be a promising tool for future autologous transplantations. View Publication

UNLABELLED Multiple myeloma remains an incurable plasma cell malignancy despite the rapidly evolving treatment landscape. Chimeric antigen receptor T cells targeted against BCMA have recently shown great promise in relapsed refractory multiple myeloma; however,all patients ultimately still progress from their disease. Lack of CAR T-cell persistence,impaired T-cell fitness in autologous CAR T-cell products and the presence of an immunosuppressive bone marrow (BM) microenvironment are contributory factors to treatment failure. We generated anti-BCMA CAR T cells from healthy donors (HD) and patients with multiple myeloma at different stages of disease to compare their T-cell profile,fitness,and cytotoxic activity in preclinical studies. We also used an ex vivo assay with multiple myeloma BM biopsies from distinct genomic subgroups to test the efficacy of HD-derived CAR T cells in a clinically relevant model. HD volunteers showed increased T-cell counts,higher CD4/CD8 ratio,and expanded na{\{i}}ve T-cell population compared with patients with multiple myeloma. After anti-BCMA CAR T-cell production patients with relapsed multiple myeloma had lower frequencies of CAR+ T cells decreased central memory phenotype and increased checkpoint inhibitory markers compared with HD-derived products which compromised their expansion and cytotoxicity against multiple myeloma cells in vitro. Importantly HD-derived CAR T cells efficiently killed primary multiple myeloma cells within the BM microenvironment of different multiple myeloma genomic subgroups and their cytotoxic activity could be boosted with gamma secretase inhibitors. In conclusion allogeneic anti-BCMA CAR T cells are a potential therapeutic strategy for patients with relapsed multiple myeloma and should be further developed in the clinic. SIGNIFICANCE Multiple myeloma is an incurable cancer of the plasma cells. A new therapy with anti-BCMA CAR T cells - the patient's own T cells genetically engineered to find and kill myeloma cancer cells - has shown encouraging results. Unfortunately patients still relapse. In this study we propose to use T cells from HD volunteers which have a stronger T-cell fitness higher cancer killing capacity and are ready to be administered when needed." View Publication

DNA repair capacity of eukaryotic cells has been studied extensively in recent years. Mammalian cells have been engineered to overexpress recombinant nuclear DNA repair proteins from ectopic genes to assess the impact of increased DNA repair capacity on genome stability. This approach has been used in this study to specifically target O(6)-methylguanine DNA methyltransferase (MGMT) to the mitochondria and examine its impact on cell survival after exposure to DNA alkylating agents. Survival of human hematopoietic cell lines and primary hematopoietic CD34(+) committed progenitor cells was monitored because the baseline repair capacity for alkylation-induced DNA damage is typically low due to insufficient expression of MGMT. Increased DNA repair capacity was observed when K562 cells were transfected with nuclear-targeted MGMT (nucl-MGMT) or mitochondrial-targeted MGMT (mito-MGMT). Furthermore,overexpression of mito-MGMT provided greater resistance to cell killing by 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) than overexpression of nucl-MGMT. Simultaneous overexpression of mito-MGMT and nucl-MGMT did not enhance the resistance provided by mito-MGMT alone. Overexpression of either mito-MGMT or nucl-MGMT also conferred a similar level of resistance to methyl methanesulfonate (MMS) and temozolomide (TMZ) but simultaneous overexpression in both cellular compartments was neither additive nor synergistic. When human CD34(+) cells were infected with oncoretroviral vectors that targeted O(6)-benzylguanine (6BG)-resistant MGMT (MGMT(P140K)) to the nucleus or the mitochondria,committed progenitors derived from infected cells were resistant to 6BG/BCNU or 6BG/TMZ. These studies indicate that mitochondrial or nuclear targeting of MGMT protects hematopoietic cells against cell killing by BCNU,TMZ,and MMS,which is consistent with the possibility that mitochondrial DNA damage and nuclear DNA damage contribute equally to alkylating agent-induced cell killing during chemotherapy. View Publication

In this study,we trace developmental stages using epigenome changes in human embryonic stem cells (hESCs) treated with drugs modulating either self-renewal or differentiation. Based on microscopy,qPCR and flow cytometry,we classified the treatment outcome as inducing pluripotency (hESC,flurbiprofen and gatifloxacin),mesendoderm (sinomenine),differentiation (cyamarin,digoxin,digitoxin,selegeline and theanine) and lineage-commitment (RA). When we analyzed histone PTMs that imprinted these gene and protein expressions,the above classification was reassorted. Hyperacetylation at H3K4,9,14,18,56 and 122 as well as H4K5,8,12 and 16 emerged as the pluripotency signature of hESCs. Methylations especially of H3 at K9,K20,K27 and K36 characterized differentiation initiation as seen in no-drug control and fluribiprofen. Sinomenine-treated cells clustered close to differentiation initiators"� View Publication

Human pluripotent stem cells (PSCs) exist in naive and primed states and provide important models to investigate the earliest stages of human development. Naive cells can be obtained through primed-to-naive resetting,but there are no reliable methods to prospectively isolate unmodified naive cells during this process. Here we report comprehensive profiling of cell surface proteins by flow cytometry in naive and primed human PSCs. Several naive-specific,but not primed-specific,proteins were also expressed by pluripotent cells in the human preimplantation embryo. The upregulation of naive-specific cell surface proteins during primed-to-naive resetting enabled the isolation and characterization of live naive cells and intermediate cell populations. This analysis revealed distinct transcriptional and X chromosome inactivation changes associated with the early and late stages of naive cell formation. Thus,identification of state-specific proteins provides a robust set of molecular markers to define the human PSC state and allows new insights into the molecular events leading to naive cell resetting. View Publication

To better understand transcriptional regulation during human oogenesis and preimplantation development,we defined stage-specific transcription,which highlighted the cleavage stage as being highly distinctive. Here,we present multiple lines of evidence that a eutherian-specific multicopy retrogene,DUX4,encodes a transcription factor that activates hundreds of endogenous genes (for example,ZSCAN4,KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably,mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells,measured here by the reactivation of '2C' genes and repeat elements,the loss of POU5F1 (also known as OCT4) protein and chromocenters,and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus,we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state. View Publication