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

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

During hematopoietic differentiation of human embryonic stem cells (hESCs),early hematopoietic progenitors arise along with endothelial cells within the CD34(+) population. Although hESC-derived hematopoietic progenitors have been previously identified by functional assays,their phenotype has not been defined. Here,using hESC differentiation in coculture with OP9 stromal cells,we demonstrate that early progenitors committed to hematopoietic development could be identified by surface expression of leukosialin (CD43). CD43 was detected on all types of emerging clonogenic progenitors before expression of CD45,persisted on differentiating hematopoietic cells,and reliably separated the hematopoietic CD34(+) population from CD34(+)CD43(-)CD31(+)KDR(+) endothelial and CD34(+)CD43(-)CD31(-)KDR(-) mesenchymal cells. Furthermore,we demonstrated that the first-appearing CD34(+)CD43(+)CD235a(+)CD41a(+/-)CD45(-) cells represent precommitted erythro-megakaryocytic progenitors. Multipotent lymphohematopoietic progenitors were generated later as CD34(+)CD43(+)CD41a(-)CD235a(-)CD45(-) cells. These cells were negative for lineage-specific markers (Lin(-)),expressed KDR,VE-cadherin,and CD105 endothelial proteins,and expressed GATA-2,GATA-3,RUNX1,C-MYB transcription factors that typify initial stages of definitive hematopoiesis originating from endothelial-like precursors. Acquisition of CD45 expression by CD34(+)CD43(+)CD45(-)Lin(-) cells was associated with progressive myeloid commitment and a decrease of B-lymphoid potential. CD34(+)CD43(+)CD45(+)Lin(-) cells were largely devoid of VE-cadherin and KDR expression and had a distinct FLT3(high)GATA3(low)RUNX1(low)PU1(high)MPO(high)IL7RA(high) gene expression profile. View Publication

Recently it has been demonstrated that CD30 expression was rather specific for transformed than for normal human ES cells and therefore CD30 maybe suggested as a potential marker for human ES cells bearing chromosomal abnormalities. Using immunohistochemistry and RT-PCR analysis we examined �?¡D30 expression in 10 hESCs lines with normal and abberant karyotypes. All hESC lines expressed CD30 antigen and RNA in undifferentiated state whether cell line beared chromosomal abnormalities or not. In contrast to previous notions our data demonstrate that CD30 could be considered as marker of undifferentiated hESCs without respect to karyotype changes. View Publication

Interferon regulatory factor 3 (IRF-3) is essential for innate intracellular immune defenses that limit virus replication,but these defenses fail to suppress human immunodeficiency virus (HIV) infection,which can ultimately associate with opportunistic coinfections and the progression to AIDS. Here,we examined antiviral defenses in CD4+ cells during virus infection and coinfection,revealing that HIV type 1 (HIV-1) directs a global disruption of innate immune signaling and supports a coinfection model through suppression of IRF-3. T cells responded to paramyxovirus infection to activate IRF-3 and interferon-stimulated gene expression,but they failed to mount a response against HIV-1. The lack of response associated with a marked depletion of IRF-3 but not IRF-7 in HIV-1-infected cells,which supported robust viral replication,whereas ectopic expression of active IRF-3 suppressed HIV-1 infection. IRF-3 depletion was dependent on a productive HIV-1 replication cycle and caused the specific disruption of Toll-like receptor and RIG-I-like receptor innate immune signaling that rendered cells permissive to secondary virus infection. IRF-3 levels were reduced in vivo within CD4+ T cells from patients with acute HIV-1 infection but not from long-term nonprogressors. Our results indicate that viral suppression of IRF-3 promotes HIV-1 infection by disrupting IRF-3-dependent signaling pathways and innate antiviral defenses of the host cell. IRF-3 may direct an innate antiviral response that regulates HIV-1 replication and viral set point while governing susceptibility to opportunistic virus coinfections. View Publication

Mesenchymal stem cells (MSCs) have been isolated from various mesodermal and ectodermal tissues. While the phenotypic and functional heterogeneity of MSCs stemming from their developmental origins has been acknowledged,the genetic and environmental factors underpinning these differences are not well-understood. Here,we investigated whether substrate stiffness mediated mechanical cues can directly modulate the development of ectodermal MSCs (eMSCs) from a precursor human neural crest stem cell (NCSC) population. We showed that NCSC-derived eMSCs were transcriptionally and functionally distinct from mesodermal bone marrow MSCs. eMSCs derived on lower substrate stiffness specifically increased their expression of the MSC marker,CD44 in a Rho-ROCK signaling dependent manner,which resulted in a concomitant increase in the eMSCs' adipogenic and chondrogenic differentiation potential. This mechanically-induced effect can only be maintained for short-term upon switching back to a stiff substrate but can be sustained for longer-term when the eMSCs were exclusively maintained on soft substrates. We also discovered that CD44 expression modulated eMSC self-renewal and multipotency via the downregulation of downstream platelet-derived growth factor receptor beta (PDGFRbeta$) signaling. This is the first instance demonstrating that substrate stiffness not only influences the differentiation trajectories of MSCs but also their derivation from upstream progenitors,such as NCSCs. View Publication

Hematopoietic stem cells (HSCs) are enriched for aldehyde dehydrogenase (ALDH) activity and ALDH is a selectable marker for human HSCs. However,the function of ALDH in HSC biology is not well understood. We sought to determine the function of ALDH in regulating HSC fate. Pharmacologic inhibition of ALDH with diethylaminobenzaldehyde (DEAB) impeded the differentiation of murine CD34(-)c-kit(+)Sca-1(+)lineage(-) (34(-)KSL) HSCs in culture and facilitated a ninefold expansion of cells capable of radioprotecting lethally irradiated mice compared to input 34(-)KSL cells. Treatment of bone marrow (BM) 34(-)KSL cells with DEAB caused a fourfold increase in 4-week competitive repopulating units,verifying the amplification of short-term HSCs (ST-HSCs) in response to ALDH inhibition. Targeted siRNA of ALDH1a1 in BM HSCs caused a comparable expansion of radioprotective progenitor cells in culture compared to DEAB treatment,confirming that ALDH1a1 was the target of DEAB inhibition. The addition of all trans retinoic acid blocked DEAB-mediated expansion of ST-HSCs in culture,suggesting that ALDH1a1 regulates HSC differentiation via augmentation of retinoid signaling. Pharmacologic inhibition of ALDH has therapeutic potential as a means to amplify ST-HSCs for transplantation purposes. View Publication

Engineered heart tissues made from human pluripotent stem cell-derived cardiomyocytes have been used for modeling cardiac pathologies,screening new therapeutics,and providing replacement cardiac tissue. Current methods measure the functional performance of engineered heart tissue by their twitch force and beating frequency,typically obtained by optical measurements. In this article,we describe a novel method for assessing twitch force and beating frequency of engineered heart tissue using magnetic field sensing,which enables multiple tissues to be measured simultaneously. The tissues are formed as thin structures suspended between two silicone posts,where one post is rigid and another is flexible and contains an embedded magnet. When the tissue contracts it causes the flexible post to bend in proportion to its twitch force. We measured the bending of the post using giant magnetoresistive (GMR) sensors located underneath a 24-well plate containing the tissues. We validated the accuracy of the readings from the GMR sensors against optical measurements. We demonstrated the utility and sensitivity of our approach by testing the effects of three concentrations of isoproterenol and verapamil on twitch force and beating frequency in real-time,parallel experiments. This system should be scalable beyond the 24-well format,enabling greater automation in assessing the contractile function of cardiomyocytes in a tissue-engineered environment. View Publication

Stem cell-based tissue engineering is a promising technology in the effort to create functional tissues of choice. To establish an efficient approach for generating hematopoietic cell lineages directly from embryonic stem (ES) cells and to study the effects of three-dimensional (3D) biomaterials on ES cell differentiation,we cultured mouse ES cells on 3D,highly porous,biomimetic scaffolds. Cell differentiation was evaluated by microscopy and flow cytometry analysis with a variety of hematopoiesis- specific markers. Our data indicate that ES cells differentiated on porous 3D scaffold structures developed embryoid bodies (EBs) similar to those in traditional two-dimensional (2D) cultures; however,unlike 2D differentiation,these EBs integrated with the scaffold and appeared embedded in a network of extracellular matrix. Most significantly,the efficiency of hematopoietic precursor cell (HPC) generation on 3D,as indicated by the expression of various HPC-specific surface markers (CD34,Sca-1,Flk-1,and c-Kit) and colony-forming cell (CFC) assays,was reproducibly increased (about 2-fold) over their 2D counterparts. Comparison of static and dynamic 3D cultures demonstrated that spinner flask technology also contributed to the higher hematopoietic differentiation efficiency of ES cells seeded on scaffolds. Continued differentiation of 3D-derived HPCs into the myeloid lineage demonstrated increased efficiency (2-fold) of generating myeloid compared with differentiation from 2D-derived HPCs. View Publication

The mechanisms that regulate hematopoietic stem cell (HSC) fate decisions between proliferation and multilineage differentiation are unclear. Members of the Wnt family of ligands that activate the canonical Wnt signaling pathway,which utilizes beta-catenin to relay the signal,have been demonstrated to regulate HSC function. In this study,we examined the role of noncanonical Wnt signaling in regulating HSC fate. We observed that noncanonical Wnt5a inhibited Wnt3a-mediated canonical Wnt signaling in HSCs and suppressed Wnt3a-mediated alterations in gene expression associated with HSC differentiation,such as increased expression of myc. Wnt5a increased short- and long-term HSC repopulation by maintaining HSCs in a quiescent G(0) state. From these data,we propose that Wnt5a regulates hematopoiesis by the antagonism of the canonical Wnt pathway,resulting in a pool of quiescent HSCs. View Publication

Hematopoiesis is tightly controlled by transcription regulatory networks,but how and when specific transcription factors control lineage commitment are still largely unknown. Within the hematopoietic stem cell (Lin(-)Sca-1(+)c-Kit(+)) compartment these lineage-specific transcription factors are expressed at low levels but are up-regulated with the process of lineage specification. CCAAT/enhancer binding protein α (C/EBPα) represents one of these factors and is involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor cells,which express C/EBPα,we developed a mouse model expressing Cre recombinase from the Cebpa promoter and a conditional EYFP allele. We show that Cebpa/EYFP(+) cells represent a significant subset of multipotent hematopoietic progenitors,which predominantly give rise to myeloid cells in steady-state hematopoiesis. C/EBPα induced a strong myeloid gene expression signature and down-regulated E2A-induced regulators of early lymphoid development. In addition,Cebpa/EYFP(+) cells compose a fraction of early thymic progenitors with robust myeloid potential. However,Cebpa/EYFP(+) multipotent hematopoietic progenitors and early thymic progenitors retained the ability to develop into erythroid and T-lymphoid lineages,respectively. These findings support an instructive but argue against a lineage-restrictive role of C/EBPα in multipotent hematopoietic and thymic progenitors. View Publication