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

Nanog,Oct4,and Sox2 are the core regulators of mouse (m)ESC pluripotency. Although their basic importance in human (h)ESCs has been demonstrated,the mechanistic functions are not well defined. Here,we identify general and cell-line-specific requirements for NANOG,OCT4,and SOX2 in hESCs. We show that OCT4 regulates,and interacts with,the BMP4 pathway to specify four developmental fates. High levels of OCT4 enable self-renewal in the absence of BMP4 but specify mesendoderm in the presence of BMP4. Low levels of OCT4 induce embryonic ectoderm differentiation in the absence of BMP4 but specify extraembryonic lineages in the presence of BMP4. NANOG represses embryonic ectoderm differentiation but has little effect on other lineages,whereas SOX2 and SOX3 are redundant and repress mesendoderm differentiation. Thus,instead of being panrepressors of differentiation,each factor controls specific cell fates. Our study revises the view of how self-renewal is orchestrated in hESCs. View Publication

Allogeneic cell therapies,such as those involving macrophages or Natural Killer (NK) cells,are of increasing interest for cancer immunotherapy. However,the current techniques for genetically modifying these cell types using lenti- or gamma-retroviral vectors present challenges,such as required cell pre-activation and inefficiency in transduction,which hinder the assessment of preclinical efficacy and clinical translation. In our study,we describe a novel lentiviral pseudotype based on the Koala Retrovirus (KoRV) envelope protein,which we identified based on homology to existing pseudotypes used in cell therapy. Unlike other pseudotyped viral vectors,this KoRV-based envelope demonstrates remarkable efficiency in transducing freshly isolated primary human NK cells directly from blood,as well as freshly obtained monocytes,which were differentiated to M1 macrophages as well as B cells from multiple donors,achieving up to 80% reporter gene expression within three days post-transduction. Importantly,KoRV-based transduction does not compromise the expression of crucial immune cell receptors,nor does it impair immune cell functionality,including NK cell viability,proliferation,cytotoxicity as well as phagocytosis of differentiated macrophages. Preserving immune cell functionality is pivotal for the success of cell-based therapeutics in treating various malignancies. By achieving high transduction rates of freshly isolated immune cells before expansion,our approach enables a streamlined and cost-effective automated production of off-the-shelf cell therapeutics,requiring fewer viral particles and less manufacturing steps. This breakthrough holds the potential to significantly reduce the time and resources required for producing e.g. NK cell therapeutics,expediting their availability to patients in need. Subject terms: Genetic transduction,Tumour immunology,Immunotherapy,Genetic vectors,Innate immune cells View Publication

Extracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising,natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture,human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However,hiPSC-EVs only have a moderate ability for brain delivery. Herein,we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor. An RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging. Here we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype,express pluripotency markers,and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways,compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency. Taken together,we have established stable,genetically engineered hiPSCs for producing EVs with RVG expression,offering the improved ability for brain-targeted drug delivery. The online version contains supplementary material available at 10.1186/s13287-024-03955-2. View Publication

hiPSC-derived blood–brain barrier (BBB) models are valuable for pharmacological and physiological studies,yet their translational potential is limited due to insufficient cell phenotypes and the neglection of the complex environment of the BBB. This study evaluates the plasma compatibility with hiPSC-derived microvascular endothelial-like cells to enhance the translational potential of in vitro BBB models. Therefore,plasma samples (sodium/lithium heparin,citrate,EDTA) and serum from healthy donors were tested on hiPSC-derived microvascular endothelial-like cells at concentrations of 100%,75%,and 50%. After 24 h,cell viability parameters were assessed. The impact of heparin-anticoagulated plasmas was further evaluated regarding barrier function and endothelial phenotype of differentiated endothelial-like cells. Finally,sodium-heparin plasma was tested in an isogenic triple-culture BBB model with continuous TEER measurements for 72 h. Only the application of heparin-anticoagulated plasmas did not significantly alter viability parameters compared to medium. Furthermore,heparin plasmas improved barrier function without increasing cell density and induced a von Willebrand factor signal. Finally,continuous TEER measurements of the triple-culture model confirmed the positive impact of sodium-heparin plasma on barrier function. Consequently,heparin-anticoagulated plasmas were proven to be compatible with hiPSC-derived microvascular endothelial-like cells. Thereby,the translational potential of BBB models can be substantially improved in the future. View Publication

Osteosarcoma (OSA) is the most common primary bone tumor in children and adolescents,yet outcomes have remained largely unchanged for over 40 years. While chimeric antigen receptor (CAR) T cell therapy has shown success in blood cancers,it faces major limitations in solid tumors due to immune evasion,antigen loss,and immunosuppressive tumor microenvironments. Natural killer (NK) cells offer several advantages over T cells,including multiple killing mechanisms and lower risks of graft-versus-host disease,neurotoxicity,and cytokine release syndrome,making them promising candidates for off-the-shelf cell therapies. However,unmodified NK cells have shown limited efficacy in clinical settings due to poor engraftment,persistence,and tumor-mediated suppression. To overcome these barriers,we developed a cost-effective method to engineer CAR NK cells targeting CD70,a tumor antigen overexpressed in relapsed and metastatic OSA. We further enhanced these cells by incorporating soluble interleukin-15 (IL-15) and a dominant-negative TGF-β receptor,creating “armored” CAR NK cells. These engineered cells resist transforming growth factor β (TGF-β) suppression,secrete IL-15,and demonstrate improved cytotoxicity,persistence,and tumor homing in both in vitro and in vivo models. Our findings support CD70 CAR NK cells as a promising immunotherapeutic strategy for relapsed and metastatic OSA. Graphical abstract Engineered “armored” CAR NK cells targeting CD70 overcome immune suppression in osteosarcoma,enhancing persistence,tumor homing,and cytotoxicity. This study presents a promising off-the-shelf immunotherapy approach for relapsed and metastatic OSA,offering a potential advance where current treatments have stagnated for decades. View Publication

PURPOSE: Preclinical in vivo studies can help guide the selection of agents and regimens for clinical testing. However,one of the challenges in screening anticancer therapies is the assessment of off-target human toxicity. There is a need for in vivo models that can simulate efficacy and toxicities of promising therapeutic regimens. For example,hematopoietic cells of human origin are particularly sensitive to a variety of chemotherapeutic regimens,but in vivo models to assess potential toxicities have not been developed. In this study,a xenograft model containing humanized bone marrow is utilized as an in vivo assay to monitor hematotoxicity. EXPERIMENTAL DESIGN: A proof-of-concept,temozolomide-based regimen was developed that inhibits tumor xenograft growth. This regimen was selected for testing because it has been previously shown to cause myelosuppression in mice and humans. The dose-intensive regimen was administered to NOD.Cg-Prkdc(scid)IL2rg(tm1Wjl)/Sz (NOD/SCID/γchain(null)),reconstituted with human hematopoietic cells,and the impact of treatment on human hematopoiesis was evaluated. RESULTS: The dose-intensive regimen resulted in significant decreases in growth of human glioblastoma xenografts. When this regimen was administered to mice containing humanized bone marrow,flow cytometric analyses indicated that the human bone marrow cells were significantly more sensitive to treatment than the murine bone marrow cells and that the regimen was highly toxic to human-derived hematopoietic cells of all lineages (progenitor,lymphoid,and myeloid). CONCLUSIONS: The humanized bone marrow xenograft model described has the potential to be used as a platform for monitoring the impact of anticancer therapies on human hematopoiesis and could lead to subsequent refinement of therapies prior to clinical evaluation. View Publication

The superiority of dendritic cells (DCs) as antigen-presenting cells has been exploited in numerous clinical trials,where generally monocyte-derived DCs (Mo-DCs) are injected to induce immunity in patients with cancer or infectious diseases. Despite promising expansion of antigen-specific T cells,the clinical responses following vaccination have been limited,indicating that further improvements of DC vaccine potency are necessary. Pre-clinical studies suggest that vaccination with combination of primary DC subsets,such as myeloid and plasmacytoid blood DCs (mDCs and pDCs,respectively),may result in stronger clinical responses. However,it is a challenge to obtain high enough numbers of primary DCs for immunotherapy,since their frequency in blood is very low. We therefore explored the possibility to generate them from hematopoietic progenitor cells (HPCs). Here,we show that by inhibiting the aryl hydrocarbon receptor with its antagonist StemRegenin 1 (SR1),clinical-scale numbers of functional BDCA2(+)BDCA4(+) pDCs,BDCA1(+) mDCs,and BDCA3(+)DNGR1(+) mDCs can be efficiently generated from human CD34(+) HPCs. The ex vivo-generated DCs were phenotypically and functionally comparable to peripheral blood DCs. They secreted high levels of pro-inflammatory cytokines such as interferon (IFN)-α,interleukin (IL)-12,and tumor necrosis factor (TNF)-α and upregulated co-stimulatory molecules and maturation markers following stimulation with Toll-like receptor (TLR) ligands. Further,they induced potent allogeneic T-cell responses and activated antigen-experienced T cells. These findings demonstrate that SR1 can be exploited to generate high numbers of functional pDCs and mDCs from CD34(+) HPCs,providing an alternative option to Mo-DCs for immunotherapy of patients with cancer or infections. View Publication

BackgroundThioredoxin-interacting protein (TXNIP) plays a role in regulating endoplasmic reticulum (ER) and oxidative stress,which disrupt glucose homeostasis in diabetes. However,the impact of TXNIP deficiency on the differentiation and functionality of human stem cell-derived somatic metabolic cells remains unclear.MethodsWe used CRISPR-Cas12a genome editing to generate TXNIP-deficient (TXNIP?/?) H1 human embryonic stem cells (H1-hESCs). These cells were differentiated into hepatocyte-like cells (HLCs) and stem-cell-derived insulin-producing islets (SC-islets). The maturation and functionality TXNIP?/? and TXNIP+/+ SC-islets were assessed by implantation under the kidney capsule of male or female NOD-SCID mice.ResultsTXNIP deficiency significantly increased H1-hESC proliferation without affecting pluripotency,viability,or differentiation potential into HLCs and SC-islets. Bulk RNA-sequencing of thapsigargin-treated TXNIP?/? and TXNIP+/+ hESCs revealed differential expression of stress-responsive genes,with enriched apoptosis-related pathways in TXNIP+/+ cells,but minimal transcriptional changes specific to TXNIP deficiency. In HLCs,TXNIP deletion reduced albumin secretion and insulin signalling,as indicated by decreased AKT phosphorylation,while showing no differences in glycolytic activity or lipid metabolism markers. Under thapsigargin-induced ER stress,TXNIP?/? HLCs exhibited transiently reduced eIF2? phosphorylation and lower BiP expression,suggesting compromised adaptive responses to prolonged stress. SC-islets derived from TXNIP?/? hESCs showed comparable viability,endocrine cell composition,and cytokine responses to TXNIP+/+ islets. Following IFN? or IFN? treatment,STAT1 phosphorylation was increased in TXNIP?/? SC-islets,indicating that IFN signalling remained intact despite TXNIP deficiency. Upon implantation into NOD-SCID mice,both TXNIP?/? and TXNIP+/+ SC-islets produced human C-peptide and responded to glucose stimulation. However,TXNIP?/? SC-islets did not demonstrate enhanced glycaemic control or glucose-stimulated insulin secretion compared to controls.ConclusionsOur study demonstrates that TXNIP deficiency does not improve the differentiation or functionality of HLCs and SC-islets. We present the generation and characterisation of TXNIP?/? and TXNIP+/+ H1-hESCs,HLCs,and SC-islets as valuable models for future studies on the role of TXNIP in metabolic cell biology.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04314-5. View Publication

The development of embryonic stem cell (ESC) therapies requires the establishment of efficient methods to differentiate ESCs into specific cell lineages. Here,we report the in vitro differentiation of common marmoset (CM) (Callithrix jacchus) ESCs into hematopoietic cells after exogenous gene transfer using vesicular stomatitis virus-glycoprotein-pseudotyped lentiviral vectors. We transduced hematopoietic genes,including tal1/scl,gata1,gata2,hoxB4,and lhx2,into CM ESCs. By immunochemical and morphological analyses,we demonstrated that overexpression of tal1/scl,but not the remaining genes,dramatically increased hematopoiesis of CM ESCs,resulting in multiple blood-cell lineages. Furthermore,flow cytometric analysis demonstrated that CD34,a hematopoietic stem/progenitor cell marker,was highly expressed in tal1/scl-overexpressing embryoid body cells. Similar results were obtained from three independent CM ESC lines. These results suggest that transduction of exogenous tal1/scl cDNA into ESCs is a promising method to induce the efficient differentiation of CM ESCs into hematopoietic stem/progenitor cells. 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

Nano- and microscale topographical cues play critical roles in the induction and maintenance of various cellular functions,including morphology,adhesion,gene regulation,and communication. Recent studies indicate that structure and function at the heart tissue level is exquisitely sensitive to mechanical cues at the nano-scale as well as at the microscale level. Although fabrication methods exist for generating topographical features for cell culture,current techniques,especially those with nanoscale resolution,are typically complex,prohibitively expensive,and not accessible to most biology laboratories. Here,we present a tunable culture platform comprised of biomimetic wrinkles that simulate the heart's complex anisotropic and multiscale architecture for facile and robust cardiac cell alignment. We demonstrate the cellular and subcellular alignment of both neonatal mouse cardiomyocytes as well as those derived from human embryonic stem cells. By mimicking the fibrillar network of the extracellular matrix,this system enables monitoring of protein localization in real time and therefore the high-resolution study of phenotypic and physiologic responses to in-vivo like topographical cues. View Publication