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

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knock down that might affect the proper silencing of these genes during differentiation. View Publication

A major road-block in stem cell therapy is the poor homing and integration of transplanted stem cells with the targeted host tissue. Human induced pluripotent stem (hiPS) cells are considered an excellent alternative to embryonic stem (ES) cells and we tested the feasibility of using small,physiological electric fields (EFs) to guide hiPS cells to their target. Applied EFs stimulated and guided migration of cultured hiPS cells toward the anode,with a stimulation threshold of textless30 mV/mm; in three-dimensional (3D) culture hiPS cells remained stationary,whereas in an applied EF they migrated directionally. This is of significance as the therapeutic use of hiPS cells occurs in a 3D environment. EF exposure did not alter expression of the pluripotency markers SSEA-4 and Oct-4 in hiPS cells. We compared EF-directed migration (galvanotaxis) of hiPS cells and hES cells and found that hiPS cells showed greater sensitivity and directedness than those of hES cells in an EF,while hES cells migrated toward cathode. Rho-kinase (ROCK) inhibition,a method to aid expansion and survival of stem cells,significantly increased the motility,but reduced directionality of iPS cells in an EF by 70-80%. Thus,our study has revealed that physiological EF is an effective guidance cue for the migration of hiPS cells in either 2D or 3D environments and that will occur in a ROCK-dependent manner. Our current finding may lead to techniques for applying EFs in vivo to guide migration of transplanted stem cells. View Publication

Avian species are important model animals for developmental biology and disease research. However,unlike in mice,where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function,clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs),the first nonmammalian iPSCs,which were clonally isolated and propagated,important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes,indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes,oligodendrocytes,and neurons. Ultimately,qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers,extraembryonic tissues,and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals. View Publication

Embryonic stem cells (ESCs) are unique cells,which have the ability to differentiate into all cell types that comprise the adult organism. Furthermore,ESCs can infinitely self-renew under optimized conditions. These features place human ESCs (hESCs) in a position where these cells can be exploited for tissue engineering and regenerative medicine approaches in treating human degenerative disorders. However,cell therapy approaches will require large amounts of clinically useable cells,not typically achievable using standard static cell culture methods. Here,we describe a method wherein clinically relevant numbers of hESCs can be generated in a cost and time effective manner. View Publication

Economic development has become a prominent issue for state governments. Nevertheless,states vary in the economic policies they choose. Two general approaches to the issue are discussed: the maintenance/attraction strategy and the creation strategy. Factor analysis allows us to gauge state effort on these two criteria. Regression analysis shows that political culture is an important factor in predicting which approach a state chooses,with traditionalistic states favoring the maintenance/attraction strategy,and moralistic states favoring the creation alternative. Other predictors of state policy choices include the condition of the economy and the diffusion of innovations. Also discussed is the interaction of political culture with other relevant variables in shaping state policies. View Publication

Regulatory T cells (Treg) play an important role in regulating immune homeostasis in health and disease. Traditionally their suppressive function has been assayed by mixing purified cell populations,which does not provide an accurate picture of a physiologically relevant response. To overcome this limitation,we here develop ‘single cell suppression profiling of human Tregs’ (scSPOT). scSPOT uses a 52-marker CyTOF panel,a cell division detection algorithm,and a whole PBMC system to assess the effect of Tregs on all other cell types simultaneously. In this head-to-head comparison,we find Tregs having the clearest suppressive effects on effector memory CD8 T cells through partial division arrest,cell cycle inhibition,and effector molecule downregulation. Additionally,scSPOT identifies a Treg phenotypic split previously observed in viral infection and propose modes of action by the FDA-approved drugs Ipilimumab and Tazemetostat. scSPOT is thus scalable,robust,widely applicable,and may be used to better understand Treg immunobiology and screen for therapeutic compounds. Traditional regulatory T cell (Tregs) assays utilize mixture of purified cell population. Here the authors develop a ‘single cell suppression profiling of human Tregs’ (scSPOT) with 52-marker CyTOF panel,a cell division detection algorithm,and a whole PBMC system to assess Treg suppressive function on all cell types simultaneously. View Publication

Highlights•Preformation of aggregates tuned by cell density enable cultivation of hiPSCs in scale-down shear environments.•Scale-down systems utilizing preformation protocols achieve comparable fold expansion with commercial systems.•Expression of pluripotency markers and functional differentiation capacity is maintained following passage in scale-down culture.•Successful application of hiPSC protocols at < 20 mL scales enable rapid and cost-effective research into cell phenotype under dynamic conditions. Human induced pluripotent stem cell (hiPSC) derived therapeutics require clinically relevant quantities of high-quality cell populations for applications in regenerative medicine. The lack of efficacy exhibited across clinical trials suggests deeper understanding of the networks governing phenotype is needed. Further,costs limit study throughput in characterizing the artificial niche relative to outcomes. We present herein an optimized strategy to enable high-throughput hiPSC expansion at <20 mL research scale. We assessed viability of single cell inoculation and aggregate preformation to facilitate proliferation. We modeled aggregate characteristics against agitation rate. Our results demonstrate tunable control with fold expansion comparable to commercial systems. Marker quantification and teratoma assay confirm functional pluripotency. This approach constitutes a scalable protocol to accelerate hiPSC research,and a significant step in advancing the rate of progress in elucidating links to derivative functionality. This work will enable statistically rigorous studies targeting hiPSC and downstream phenotype for clinical manufacturing. Graphical abstractImplementation of adapted protocols enable scale-down systems as a tool for high-throughput iPSC biomanufacturing research,in platforms conducive to scale-up for clinical manufacturing.Image,graphical abstract View Publication

Three-dimensional (3D) human brain tissue models derived from induced pluripotent stem cells (iPSCs) have transformed the study of neural development and disease in vitro. While cerebral organoids offer high structural complexity,their large size often leads to necrotic core formation,limiting reproducibility and challenging the integration of microglia. Here,we present a detailed,reproducible protocol for generating multi-cell type 3D neurospheres that incorporate neurons,astrocytes,and optionally microglia,all derived from the same iPSCs. While neurons and astrocytes differentiate spontaneously from neural precursor cells,generated by dual SMAD-inhibition (blocking BMP and TGF-b signaling),microglia are generated in parallel and can infiltrate the mature neurosphere tissue after plating neurospheres into 48-well plates. The system supports a range of downstream applications,including functional confocal live imaging of GCaMP6f after adeno-associated virus (AAV) transduction of neurospheres or immunofluorescence staining after fixation. Our approach has been successfully implemented across multiple laboratories,demonstrating its robustness and translational potential for studying neuron–glia interactions and modeling neurodegenerative processes. View Publication

Epstein-Barr virus (EBV) causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies,particularly in immune-compromised hosts. Transplant patients face high risk of post-transplant lymphoproliferative disease,a life-threatening EBV-driven lymphoma. There are no EBV-specific vaccines or treatments; however,neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents. EBV entry into B cells involves gp350,which binds complement receptors,and gp42,which engages HLA class II to trigger fusion. Most existing monoclonal antibodies (mAbs) against these antigens are non-human,limiting clinical use. Using a transgenic mouse model,we generate two gp350 and eight gp42 genetically human neutralizing mAbs that block receptor binding. Structural analyses reveal extended sites of vulnerability relevant to vaccine development. Delivery of a gp42 mAb protects humanized mice from EBV challenge,while a gp350 mAb provides partial protection. These mAbs highlight the utility of transgenic mice to produce therapeutic mAbs for preventing EBV-driven disease. Graphical abstract Highlights•Transgenic mice were used to make genetically human EBV mAbs against gp350 and gp42•mAbs potently neutralize EBV infection by blocking receptor-ligand interactions•mAbs prevent EBV infection following EBV challenge in humanized mice Epstein-Barr virus (EBV) can cause serious illness,including cancer,especially in immunocompromised patients. There are no EBV-specific treatments. Chhan et al. leverage a transgenic mouse model to develop human monoclonal antibodies that block EBV entry. These antibodies prevent EBV infection in a murine challenge model offering hope for new therapies. View Publication

We present a predictive bioprocess design strategy employing cell- and molecular-level analysis of rate-limiting steps in human pluripotent stem cell (hPSC) expansion and differentiation,and apply it to produce definitive endoderm (DE) progenitors using a scalable directed-differentiation technology. We define a bioprocess optimization parameter (L; targeted cell Loss) and,with quantitative cell division tracking and fate monitoring,identify and overcome key suspension bioprocess bottlenecks. Adapting process operating conditions to pivotal parameters (single cell survival and growth rate) in a cell-line-specific manner enabled adherent-equivalent expansion of hPSCs in feeder- and matrix-free defined-medium suspension culture. Predominantly instructive differentiation mechanisms were found to underlie a subsequent 18-fold expansion,during directed differentiation,to high-purity DE competent for further commitment along pancreatic and hepatic lineages. This study demonstrates that iPSC expansion and differentiation conditions can be prospectively specified to guide the enhanced production of target cells in a scale-free directed differentiation system. View Publication

Multiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting G(αs)-,G(α-i/o)- and G(α-q/11)-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ,intracellular free calcium and CAMKII kinase activity downstream of G(α-q/11) as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a G(α-q/11) subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly,treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs,lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (ptextless0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture. View Publication

Over 4% of the global population is estimated to live with autoimmune disease,necessitating immunosuppressive treatment that is often chronic,not curative,and carries associated risks. B cells have emerged as key players in disease pathogenesis,as evidenced by partial responsiveness to B cell depletion by antibody-based therapies. However,these treatments often have transient effects due to incomplete depletion of tissue-resident B cells. Chimeric antigen receptor (CAR) T cells targeting B cells have demonstrated efficacy in refractory systemic lupus erythematosus. To this end,we developed an anti-CD19 CAR T cell product candidate,CABA-201,containing a clinically evaluated fully human CD19 binder (IC78) with a 4-1BB costimulatory domain and CD3 zeta stimulation domain for treatment refractory autoimmune disease. Here,we demonstrate specific cytotoxic activity of CABA-201 against CD19+ Nalm6 cells with no off-target effects on primary human cells. Novel examination of CABA-201 generated from primary T cells from multiple patients with autoimmune disease displayed robust CAR surface expression and effective elimination of the intended target autologous CD19+ B cells in vitro. Together,these findings support the tolerability and activity of CABA-201 for clinical development in patients with autoimmune disease. Graphical abstract Basu and colleagues show CABA-201,a B cell-targeting CAR T cell product with a fully human CD19 binder and 4-1BB costimulatory domain,can precisely eliminate autoimmune patient B cells without off-target deleterious effects,demonstrating its ability as a robust therapeutic for B cell-driven autoimmune disorders. View Publication