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

Many human embryonic stem (hES) and induced pluripotent stem (hiPS) cell differentiation protocols begin with the formation of three-dimensional aggregates of cells called embryoid bodies (EBs). Traditional EB formation methods result in a heterogeneous population of EB sizes and shapes,which then undergo heterogeneous differentiation efficiencies. AggreWell(TM)400 and AggreWell(TM)800 use the spin-EB method to force the aggregation of a defined number of cells,thereby controlling EB size and generating a population of uniform EBs. Moreover,the dense array of microwells on the bottom surface of AggreWell(TM)400 provide for the rapid and simple production of thousands of EBs at a time. View Publication

Teratoma formation is a critical obstacle to safe clinical translation of human embryonic stem (ES) cell-based therapies in the future. As current methods of isolation are unable to yield 100% pure population of differentiated cells from a pluripotent donor source,potential development of these tumors is a significant concern. Here we used non-invasive reporter gene imaging to investigate the relationship between human ES cell number and teratoma formation in a xenogenic model of ES cell transplantation. Human ES cells (H9 line) were stably transduced with a double fusion (DF) reporter construct containing firefly luciferase and enhanced green fluorescent protein (Fluc- eGFP) driven by a human ubiquitin promoter. Immunodeficient mice received intramyocardial (n = 35) or skeletal muscle (n = 35) injection of 1 × 102,1 × 103,1 × 104,1 × 105 or 1 × 106 DF positive ES cells suspended in saline for myocardium and Matrigel for skeletal muscle. Cell survival and proliferation were monitored via bioluminescence imaging (BLI) for an 8 week period following transplantation. Mice negative for Fluc signal after 8 weeks were followed out to day 365 to confirm tumor absence. Significantly,in this study,a minimum of 1 × 105 ES cells in the myocardium and 1 × 104 cells in the skeletal muscle was observed to be requisite for teratoma development,suggesting that human ES cell number may be a critical factor in teratoma formation. Engraftment and tumor occurrence were also observed to be highly dependent on ES cell number. We anticipate these results should yield useful insights to the safe and reliable application of human ES cell derivatives in the clinic. Keywords View Publication

We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts,an apical domain,marked by EZRIN and atypical PKC$\$,is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly,actin polymerization,regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA),promotes lumen formation,whereas actin contraction,mediated by MYOSIN-II,inhibits this process. Finally,we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development,this system can provide a powerful model for investigation of this process in a controlled environment. Overall,our data establish that lumenogenesis is a fundamental cell biological property of human PSCs. View Publication

BACKGROUND: Many patients with ischemic heart disease have cardiovascular risk factors such as cigarette smoking. We tested the effect of nicotine (a key component of cigarette smoking) on the therapeutic effects of human embryonic stem cell-derived endothelial cells (hESC-ECs).backslashnbackslashnMETHODS AND RESULTS: To induce endothelial cell differentiation,undifferentiated hESCs (H9 line) underwent 4-day floating EB formation and 8-day outgrowth differentiation in EGM-2 media. After 12 days,CD31(+) cells (13.7+/-2.5%) were sorted by FACScan and maintained in EGM-2 media for further differentiation. After isolation,these hESC-ECs expressed endothelial specific markers such as vWF (96.3+/-1.4%),CD31 (97.2+/-2.5%),and VE-cadherin (93.7+/-2.8%),form vascular-like channels,and incorporated DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL). Afterward,5x10(6) hESC-ECs treated for 24 hours with nicotine (10(-8) M) or PBS (as control) were injected into the hearts of mice undergoing LAD ligation followed by administration for two weeks of vehicle or nicotine (100 microg/ml) in the drinking water. Surprisingly,bioluminescence imaging (BLI) showed significant improvement in the survival of transplanted hESC-ECs in the nicotine treated group at 6 weeks. Postmortem analysis confirmed increased presence of small capillaries in the infarcted zones. Finally,in vitro mechanistic analysis suggests activation of the MAPK and Akt pathways following activation of nicotinic acetylcholine receptors (nAChRs).backslashnbackslashnCONCLUSIONS: This study shows for the first time that short-term systemic administrations of low dose nicotine can improve the survival of transplanted hESC-ECs,and enhance their angiogenic effects in vivo. Furthermore,activation of nAChRs has anti-apoptotic,angiogenic,and proliferative effects through MAPK and Akt signaling pathways. View Publication

Deciding to exit pluripotency and undergo differentiation is of singular importance for pluripotent cells,including embryonic stem cells (ESCs). The molecular mechanisms for these decisions to differentiate,as well as reversing those decisions during induced pluripotency (iPS),have focused largely on transcriptomic controls. Here,we explore the role of translational control for the maintenance of pluripotency and the decisions to differentiate. Global protein translation is significantly reduced in hESCs compared to their differentiated progeny. Furthermore,p70 S6K activation is restricted in hESCs compared to differentiated fibroblast-like cells. Disruption of p70 S6K-mediated translation by rapamycin or siRNA knockdown in undifferentiated hESCs does not alter cell viability or expression of the pluripotency markers Oct4 and Nanog. However,expression of constitutively active p70 S6K,but not wild-type p70 S6K,induces differentiation. Additionally,hESCs exhibit high levels of the mTORC1/p70 S6K inhibitory complex TSC1/TSC2 and preferentially express more rapamycin insensitive mTORC2 compared to differentiated cells. siRNA-mediated knockdown of both TSC2 and Rictor elevates p70 S6K activation and induces differentiation of hESCs. These results suggest that hESCs tightly regulate mTORC1/p70 S6K-mediated protein translation to maintain a pluripotent state as well as implicate a novel role for protein synthesis as a driving force behind hESC differentiation. View Publication

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have blood cells deficient in glycosyl phosphatidylinositol (GPI)-linked proteins owing to a somatic mutation in the X-linked PIGA gene. To target Piga recombination to the erythroid/megakaryocytic lineage in mice,the Cre/loxP system was used,and Cre was expressed under the transcriptional regulatory sequences of GATA-1. Breeding of GATA1-cre (G) transgenic mice with mice carrying a floxed Piga (L) allele was associated with high embryonic lethality. However,double-transgenic (GL) mice that escaped early recombination looked healthy and were observed for 16 months. Flow cytometric analysis of peripheral blood cells showed that GL mice had up to 100% of red cells deficient in GPI-linked proteins. The loss of GPI-linked proteins on the cell surface occurred late in erythroid differentiation,causing a proportion of red cells to express low residual levels of GPI-linked proteins. Red cells with residual expression of GPI-linked proteins showed an intermediate sensitivity toward complement and thus resemble PNH type II cells in patients with PNH. Recombination of the floxed Piga allele was also detected in cultured megakaryocytes,mast cells,and eosinophils,but not in neutrophils,lymphocytes,or nonhematopoietic tissues. In summary,GATA1-Cre causes high-efficiency Piga gene inactivation in a GATA-1-specific pattern. For the first time,mice were generated that have almost 100% of red cells deficient in GPI-linked proteins. These animals will be valuable to further investigate the consequences of GPI-anchor deficiency on erythroid/megakaryocytic cells. View Publication

Bone morphogenetic protein (BMP) signaling regulates embryonic hematopoiesis via receptor-mediated activation of downstream SMAD proteins,including SMAD1. In previous work,we showed that Smad1 expression is sufficient to enhance commitment of mesoderm to hemangioblast fate. We also found indirect evidence to support a subsequent repressive function for Smad1 in hematopoiesis. To test this hypothesis directly,we developed a novel system allowing temporal control of Smad1 levels by conditional knockdown in embryonic stem cell derivatives. Depletion of Smad1 in embryoid body cultures before hemangioblast commitment limits hematopoietic potential because of a block in mesoderm development. Conversely,when Smad1 is depleted in FlK1(+) mesoderm,at a stage after hemangioblast commitment,the pool of hematopoietic progenitors is expanded. This involves enhanced expression levels for genes specific to hematopoiesis,including Gata1,Runx1 and Eklf,rather than factors required for earlier specification of the hemangioblast. The phenotype correlates with increased nuclear SMAD2 activity,indicating molecular cross-regulation between the BMP and TGF-β signaling pathways. Consistent with this mechanism,hematopoiesis was enhanced when Smad2 was directly expressed during this same developmental window. Therefore,this study reveals a temporally defined function for Smad1 in restricting the expansion of early hematopoietic progenitors. View Publication

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells,there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression,we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype,including haematopoietic and stromal cells as well as a neuronal niche. Collectively,our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues. View Publication

Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent postnatal stem cells that have been used for the treatment of bone defects and graft-versus-host diseases in clinics. In this study,we found that subcutaneously transplanted human BMMSCs are capable of organizing hematopoietic progenitors of recipient origin. These hematopoietic cells expressed multiple lineages of hematopoietic cell associated markers and were able to rescue lethally irradiated mice,with successful engraftment in the recipient,suggesting a potential bone marrow (BM) resource for stem cell therapies. Furthermore,we found that platelet-derived growth factor (PDGF) promotes the formation of BMMSC-generated BM niches through upregulation of beta-catenin,implying that the PDGF pathway contributes to the formation of ectopic BM. These results indicate that the BMMSC-organized BM niche system represents a unique hematopoietic progenitor resource possessing potential clinical value. View Publication

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However,development of cell-based regenerative therapies has been hindered by the lack of preclinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into beta-glucuronidase (GUSB)-deficient NOD/SCID/mucopolysaccharidosis type VII mice,we characterized the distribution of lineage-depleted human umbilical cord blood-derived cells purified by selection using high aldehyde dehydrogenase (ALDH) activity with CD133 coexpression. ALDH(hi) or ALDH(hi)CD133+ cells produced robust hematopoietic reconstitution and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that coexpressed human leukocyte antigen (HLA-A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues,including islet and ductal regions of mouse pancreata. In contrast,variable phenotypes were detected in the chimeric liver,with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels and CD45-/HLA- cells with diluted GUSB expression predominant in the liver parenchyma. However,true nonhematopoietic human (HLA+/CD45-) cells were rarely detected in other peripheral tissues,suggesting that these GUSB+/HLA-/CD45- cells in the liver were a result of downregulated human surface marker expression in vivo,not widespread seeding of nonhematopoietic cells. However,relying solely on continued expression of cell surface markers,as used in traditional xenotransplantation models,may underestimate true tissue distribution. ALDH-expressing progenitor cells demonstrated widespread and tissue-specific distribution of variable cellular phenotypes,indicating that these adult progenitor cells should be explored in transplantation models of tissue damage. View Publication

Gamma-aminobutyric acid (GABA)-releasing interneurons play an important modulatory role in the cortex and have been implicated in multiple neurological disorders. Patient-derived interneurons could provide a foundation for studying the pathogenesis of these diseases as well as for identifying potential therapeutic targets. Here,we identified a set of genetic factors that could robustly induce human pluripotent stem cells (hPSCs) into GABAergic neurons (iGNs) with high efficiency. We demonstrated that the human iGNs express neurochemical markers and exhibit mature electrophysiological properties within 6-8 weeks. Furthermore,in vitro,iGNs could form functional synapses with other iGNs or with human-induced glutamatergic neurons (iENs). Upon transplantation into immunodeficient mice,human iGNs underwent synaptic maturation and integration into host neural circuits. Taken together,our rapid and highly efficient single-step protocol to generate iGNs may be useful to both mechanistic and translational studies of human interneurons. View Publication

Pluripotency makes human pluripotent stem cells (hPSCs) promising for regenerative medicine,but the teratoma formation has been considered to be a major obstacle for their clinical applications. Here,we determined that the downregulation of miR-302 suppresses the teratoma formation,hampers the self-renewal and pluripotency,and promotes hPSC differentiation. The underlying mechanism is that the high endogenous expression of miR-302 suppresses the AKT1 expression by directly targeting its 3'UTR and subsequently maintains the pluripotent factor OCT4 at high level. Our findings reveal that miR-302 regulates OCT4 by suppressing AKT1,which provides hPSCs two characteristics related to their potential for clinical applications: the benefit of pluripotency and the hindrance of teratoma formation. More importantly,we demonstrate that miR-302 upregulation cannot lead OCT4 negative human adult mesenchymal stem cells (hMSCs) to acquire the teratoma formation in vivo. Whether miR-302 upregulation can drive hMSCs to acquire a higher differentiation potential is worthy of deep investigation. View Publication