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

Human-induced pluripotent stem cell (iPSC)-derived mesenchymal stem cells (iMSCs) represent a promising and renewable cell source for therapeutic applications. A systematic evaluation of the immunological properties and engraftment potential of iMSCs generated from urine-derived iPSCs is lacking,which has impeded their broader application. In this study,we differentiated urine-derived iPSCs into iMSCs and assessed their fundamental MSC characteristics,immunogenicity,immunomodulatory capacity and in vivo engraftment. Compared to umbilical cord-derived MSCs (UCMSCs),iMSCs demonstrated an enhanced proliferative capacity,a higher level of regenerative gene expression,and lower immunogenicity,strengthening resistance to apoptosis induced by allogeneic peripheral blood mononuclear cells (PBMCs) and the NK-92 cell line. In addition,iMSCs exhibited a diminished ability to inhibit T cell proliferation and activation compared with UCMSCs. Transcriptomic analyses further revealed the decreased expression of immune regulatory factors in iMSCs. After transfusion into mouse models,iMSCs engrafted in the lungs,liver,and spleen and exhibited the ability to migrate to tumor tissues. Our results indicated that iMSCs generated from urine-derived iPSCs have a significant replicative capacity,low immunogenicity and unique immunomodulatory properties,and hence offer obvious advantages in immune privilege and allogenic therapeutic application prospects. View Publication

The emergence of retinal progenitor cells and differentiation to various retinal cell types represent fundamental processes during retinal development. Herein,we provide a comprehensive single cell characterisation of transcriptional and chromatin accessibility changes that underline retinal progenitor cell specification and differentiation over the course of human retinal development up to midgestation. Our lineage trajectory data demonstrate the presence of early retinal progenitors,which transit to late,and further to transient neurogenic progenitors,that give rise to all the retinal neurons. Combining single cell RNA-Seq with spatial transcriptomics of early eye samples,we demonstrate the transient presence of early retinal progenitors in the ciliary margin zone with decreasing occurrence from 8 post-conception week of human development. In retinal progenitor cells,we identified a significant enrichment for transcriptional enhanced associate domain transcription factor binding motifs,which when inhibited led to loss of cycling progenitors and retinal identity in pluripotent stem cell derived organoids. Formation of the retina during development involves the coordinated action of retinal progenitor cells and their differentiated cell types,which is key for producing a functioning eye. Here the authors provide a detailed atlas of human retinal development,combining scRNA-seq and spatial transcriptomics,and identify key genetic factors that mediate retinal progenitor cell proliferation and differentiation. View Publication

Mounting evidence indicates that exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exosomes) combine the advantages of hucMSC pluripotency with their nanoscale dimensions,enhancing their clinical potential through prolonged circulation half-life. Despite these promising characteristics,research on their immunological toxicity remains insufficient. This study focuses on the impact of hucMSC-exosomes on the general toxicity and immunopathological indicators. When mice received tail vein injections of 6 × 10 10 hucMSC-exosomes particles,we observed no significant changes in body weight,feed intake,blood composition,organ indices,or histopathological findings throughout the 14 days observation period. Similarly,blood levels of immunoglobulins,cytokines,and lymphocyte subpopulations remained stable. The hucMSC-exosomes produced no detectable negative effects on immune organs including the thymus,spleen,and bone marrow. These findings indicate that intravenous administration of 6 × 10 10 particles of hucMSC-exosomes appears relatively safe at the murine level. This assessment of safety and immunological impact following intravenous hucMSC-exosomes infusion offers experimental support for potential clinical applications and future analyses in this field. View Publication

Shifts in cellular metabolic phenotypes have the potential to cause disease-driving processes in respiratory disease. The respiratory epithelium is particularly susceptible to metabolic shifts in disease,but our understanding of these processes is limited by the incompatibility of the technology required to measure metabolism in real-time with the cell culture platforms used to generate differentiated respiratory epithelial cell types. Thus,to date,our understanding of respiratory epithelial metabolism has been restricted to that of basal epithelial cells in submerged culture,or via indirect end point metabolomics readouts in lung tissue. Here we present a novel methodology using the widely available Seahorse Analyzer platform to monitor real-time changes in the cellular metabolism of fully differentiated primary human airway epithelial cells grown at air-liquid interface (ALI). We show increased glycolytic,but not mitochondrial,ATP production rates in response to physiologically relevant increases in glucose availability. We also show that pharmacological inhibition of lactate dehydrogenase is able to reduce glucose-induced shifts toward aerobic glycolysis. This method is timely given the recent advances in our understanding of new respiratory epithelial subtypes that can only be observed in vitro through culture at ALI and will open new avenues to measure real-time metabolic changes in healthy and diseased respiratory epithelium,and in turn the potential for the development of novel therapeutics targeting metabolic-driven disease phenotypes. View Publication

A human cell-based in vitro model that can accurately predict drug penetration into the brain as well as metrics to assess these in vitro models are valuable for the development of new therapeutics. Here,human induced pluripotent stem cells (hPSCs) are differentiated into a polarized monolayer that express blood-brain barrier (BBB)-specific proteins and have transendothelial electrical resistance (TEER) values greater than 2500 Ωtextperiodcenteredcm(2). By assessing the permeabilities of several known drugs,a benchmarking system to evaluate brain permeability of drugs was established. Furthermore,relationships between TEER and permeability to both small and large molecules were established,demonstrating that different minimum TEER thresholds must be achieved to study the brain transport of these two classes of drugs. This work demonstrates that this hPSC-derived BBB model exhibits an in vivo-like phenotype,and the benchmarks established here are useful for assessing functionality of other in vitro BBB models. View Publication

Intracellular delivery of macromolecules is a challenge in research and therapeutic applications. Existing vector-based and physical methods have limitations,including their reliance on exogenous materials or electrical fields,which can lead to toxicity or off-target effects. We describe a microfluidic approach to delivery in which cells are mechanically deformed as they pass through a constriction 30–80% smaller than the cell diameter. The resulting controlled application of compression and shear forces results in the formation of transient holes that enable the diffusion of material from the surrounding buffer into the cytosol. The method has demonstrated the ability to deliver a range of material,such as carbon nanotubes,proteins,and siRNA,to 11 cell types,including embryonic stem cells and immune cells. When used for the delivery of transcription factors,the microfluidic devices produced a 10-fold improvement in colony formation relative to electroporation and cell-penetrating peptides. Indeed,its ability to deliver structurally diverse materials and its applicability to difficult-to-transfect primary cells indicate that this method could potentially enable many research and clinical applications. View Publication

BACKGROUND Microglia,the principle immune cells of the brain,play important roles in neuronal development,homeostatic function and neurodegenerative disease. Recent genetic studies have further highlighted the importance of microglia in neurodegeneration with the identification of disease risk polymorphisms in many microglial genes. To better understand the role of these genes in microglial biology and disease,we,and others,have developed methods to differentiate microglia from human induced pluripotent stem cells (iPSCs). While the development of these methods has begun to enable important new studies of microglial biology,labs with little prior stem cell experience have sometimes found it challenging to adopt these complex protocols. Therefore,we have now developed a greatly simplified approach to generate large numbers of highly pure human microglia. RESULTS iPSCs are first differentiated toward a mesodermal,hematopoietic lineage using commercially available media. Highly pure populations of non-adherent CD43+ hematopoietic progenitors are then simply transferred to media that includes three key cytokines (M-CSF,IL-34,and TGF$\beta$-1) that promote differentiation of homeostatic microglia. This updated approach avoids the prior requirement for hypoxic incubation,complex media formulation,FACS sorting,or co-culture,thereby significantly simplifying human microglial generation. To confirm that the resulting cells are equivalent to previously developed iPSC-microglia,we performed RNA-sequencing,functional testing,and transplantation studies. Our findings reveal that microglia generated via this simplified method are virtually identical to iPS-microglia produced via our previously published approach. To also determine whether a small molecule activator of TGF$\beta$ signaling (IDE1) can be used to replace recombinant TGF$\beta$1,further reducing costs,we examined growth kinetics and the transcriptome of cells differentiated with IDE1. These data demonstrate that a microglial cell can indeed be produced using this alternative approach,although transcriptional differences do occur that should be considered. CONCLUSION We anticipate that this new and greatly simplified protocol will enable many interested labs,including those with little prior stem cell or flow cytometry experience,to generate and study human iPS-microglia. By combining this method with other advances such as CRISPR-gene editing and xenotransplantation,the field will continue to improve our understanding of microglial biology and their important roles in human development,homeostasis,and disease. View Publication

The demonstration that the small synthetic molecule reversine [2-(4-morpholinoanilino)-N6-cyclohexyladenine] promotes the dedifferentiation of committed cells into multipotent progenitor-type cells has raised hopes on the exploitation of this small chemical tool for the generation of stem cells. Here,we show that reversine causes a failure in cytokinesis and induces polyploidization. These effects of reversine are due to the inhibition of Aurora A and B,two related kinases that are implicated in several aspects of mitosis and that are frequently amplified and overexpressed in human tumors. Reversine inhibits the phosphorylation of histone H3,a direct downstream target of Aurora kinases. Similarly to the Aurora kinase inhibitor VX-680,which has recently entered phase II clinical trials for cancer treatment,reversine inhibited colony formation of leukemic cells from patients with acute myeloid leukemia but was significantly less toxic than VX-680 on cells from healthy donors. The crystal structure of the reversine-Aurora B kinase complex shows that reversine is a novel class of ATP-competitive Aurora kinase inhibitors. Thus,although our studies raise serious doubts on the application of reversine in regenerative medicine,they support the paradigm that reversine might be a useful agent in cancer chemotherapy. View Publication

BACKGROUND The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects,including a reduction in cancer risk. METHODS We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations. RESULTS In vitro studies showed that cerivastatin,pitavastatin,and fluvastatin were the most potent anti-proliferative,autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently,pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment,tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells. CONCLUSIONS Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies. View Publication

Activation of ErbB4 receptor signaling is instrumental in heart development,lack of which results in embryonic lethality. However,mechanism governing its intracellular signaling remains elusive. Using human pluripotent stem cells,we show that ErbB4 is critical for cardiogenesis whereby its genetic knockdown results in loss of cardiomyocytes. Phospho-proteome profiling and Western blot studies attribute this loss to inactivation of p38$\$ isoform which physically interacts with NKx2.5 and GATA4 transcription factors. Post-cardiomyocyte formation p38$\$/NKx2.5 downregulation is followed by p38$\$/MEF2c upregulation suggesting stage-specific developmental roles of p38 MAPK isoforms. Knockdown of p38$\$ similarly disrupts cardiomyocyte formation in spite of the presence of NKx2.5. Cell fractionation and NKx2.5 phosphorylation studies suggest inhibition of ErbB4-p38$\$ hinders NKx2.5 nuclear translocation during early cardiogenesis. This study reveals a novel pathway that directly links ErbB4 and p38$\$ the transcriptional machinery of NKx2.5-GATA4 complex which is critical for cardiomyocyte formation during mammalian heart development. View Publication