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

Amniogenesis-the development of amnion-is a critical developmental milestone for early human embryogenesis and successful pregnancy. However,human amniogenesis is poorly understood due to limited accessibility to peri-implantation embryos and a lack of in vitro models. Here we report an efficient biomaterial system to generate human amnion-like tissue in vitro through self-organized development of human pluripotent stem cells (hPSCs) in a bioengineered niche mimicking the in vivo implantation environment. We show that biophysical niche factors act as a switch to toggle hPSC self-renewal versus amniogenesis under self-renewal-permissive biochemical conditions. We identify a unique molecular signature of hPSC-derived amnion-like cells and show that endogenously activated BMP-SMAD signalling is required for the amnion-like tissue development by hPSCs. This study unveils the self-organizing and mechanosensitive nature of human amniogenesis and establishes the first hPSC-based model for investigating peri-implantation human amnion development,thereby helping advance human embryology and reproductive medicine. View Publication

The identification of the trans-acting factors and cis-regulatory modules that are involved in human pluripotent stem cell (hPSC) maintenance and differentiation is necessary to dissect the operating regulatory networks in these processes and thereby identify nodes where signal input will direct desired cell fate decisions in vitro or in vivo. To deconvolute these networks,we established a method to influence the differentiation state of hPSCs with a CRISPR-associated catalytically inactive dCas9 fused to an effector domain. In human embryonic stem cells,we find that the dCas9 effectors can exert positive or negative regulation on the expression of developmentally relevant genes,which can influence cell differentiation status when impinging on a key node in the regulatory network that governs the cell state. This system provides a platform for the interrogation of the underlying regulators governing specific differentiation decisions,which can then be employed to direct cellular differentiation down desired pathways. View Publication

Tuberculosis is a leading cause of death in mankind due to infectious agents,and Mycobacterium tuberculosis (Mtb) infects and survives in macrophages (MФs). Although MФs are a major niche,myeloid-derived suppressor cells (MDSCs) are an alternative site for pathogen persistence. Both MФs and MDSCs express varying levels of leukocyte immunoglobulin-like receptor B (LILRB),which regulate the myeloid cell suppressive function. Herein,we demonstrate that antagonism of LILRB2 by a monoclonal antibody (mab) induced a switch of human MDSCs towards an M1-macrophage phenotype,increasing the killing of intracellular Mtb. Mab-mediated antagonism of LILRB2 alone and its combination with a pharmacological blockade of SHP1/2 phosphatase increased proinflammatory cytokine responses and phosphorylation of ERK1/2,p38 MAPK,and NF-kB in Mtb-infected MDSCs. LILRB2 antagonism also upregulated anti-mycobacterial iNOS gene expression and an increase in both nitric oxide and reactive oxygen species synthesis. Because genes associated with the anti-mycobacterial function of M1-MФs were enhanced in MDSCs following mab treatment,we propose that LILRB2 antagonism reprograms MDSCs from an immunosuppressive state towards a pro-inflammatory phenotype that kills Mtb. LILRB2 is therefore a novel therapeutic target for eradicating Mtb in MDSCs. View Publication

In systemic lupus erythematosus,defective clearance of apoptotic debris and activation of innate cells result in a chronically activated type 1 IFN response,which can be measured in PBMCs of most patients. Metformin,a widely used prescription drug for Type 2 diabetes,has a therapeutic effect in several mouse models of lupus through mechanisms involving inhibition of oxidative phosphorylation and a decrease in CD4+ T cell activation. In this study,we report that in CD4+ T cells from human healthy controls and human systemic lupus erythematosus patients,metformin inhibits the transcription of IFN-stimulated genes (ISGs) after IFN-alpha treatment. Accordingly,metformin inhibited the phosphorylation of pSTAT1 (Y701) and its binding to IFN-stimulated response elements that control ISG expression. These effects were independent of AMPK activation or mTORC1 inhibition but were replicated using inhibitors of the electron transport chain respiratory complexes I,III,and IV. This indicates that mitochondrial respiration is required for ISG expression in CD4+ T cells and provides a novel mechanism by which metformin may exert a therapeutic effect in autoimmune diseases. View Publication

We have developed an in vitro clonal assay of murine hematopoietic precursor cells that form spleen colonies (CFU-S day 12) or produce in vitro clonable progenitors in the marrow (MRA cells) of lethally irradiated mice. The assay is essentially a long-term bone marrow culture in microtiter wells containing marrow-derived stromal feeders" depleted for hematopoietic activity by irradiation. To test the validity of the assay as a quantitative in vitro stem cell assay� View Publication

Hematopoietic cells (HCs) promote blood vessel formation by producing various proangiogenic cytokines and chemokines and matrix metalloproteinases. We injected mouse colon26 colon cancer cells or human PC3 prostate adenocarcinoma cells into mice and studied the localization of HCs during tumor development. HCs were distributed in the inner tumor mass in all of the tumor tissues examined; however,the localization of HCs in the tumor tissue differed depending on the tumor cell type. In the case of colon26 tumors,as the tumor grew,many mature HCs migrated into the tumor mass before fine capillary formation was observed. On the other hand,although very few HCs migrated into PC3 tumor tissue,c-Kit+ hematopoietic stem/progenitor cells accumulated around the edge of the tumor. Bone marrow suppression induced by injection of anti-c-Kit neutralizing antibody suppressed tumor angiogenesis by different mechanisms according to the tumor cell type: bone marrow suppression inhibited the initiation of sprouting angiogenesis in colon26 tumors,while it suppressed an increase in the caliber of newly developed blood vessels at the tumor edge in PC3 tumors. Our findings suggest that HCs are involved in tumor angiogenesis and regulate the angiogenic switch during tumorigenesis. View Publication

Parthenogenetic embryonic stem (ES) cells with two oocyte-derived genomes (uniparental) have been proposed as a source of autologous tissue for transplantation. The therapeutic applicability of any uniparental cell type is uncertain due to the consequences of genomic imprinting that in mammalian uniparental tissues causes unbalanced expression of imprinted genes. We transplanted uniparental fetal liver cells into lethally irradiated adult mice to test their capacity to replace adult hematopoietic tissue. Both maternal (gynogenetic) and paternal (androgenetic) derived cells conveyed long-term,multilineage reconstitution of hematopoiesis in recipients,with no associated pathologies. We also establish that uniparental ES cells can differentiate into transplantable hematopoietic progenitors in vitro that contribute to long-term hematopoiesis in recipients. Hematopoietic tissue in recipients maintained fidelity of parent-of-origin methylation marks at the Igf2/H19 locus; however,variability occurred in the maintenance of parental-specific methylation marks at other loci. In summary,despite genomic imprinting and its consequences on development that are particularly evident in the androgenetic phenotype,uniparental cells of both parental origins can form adult-transplantable stem cells and can repopulate an adult organ. View Publication

INTRODUCTION: The induced pluripotent stem cell (iPSC) technology allows generation of patient-specific pluripotent stem cells,thereby providing a novel cell-therapy platform for severe degenerative diseases. One of the key issues for clinical-grade iPSC derivation is the accessibility of donor cells used for reprogramming. METHODS: We examined the feasibility of reprogramming mobilized GMP-grade hematopoietic progenitor cells (HPCs) and peripheral blood mononuclear cells (PBMCs) and tested the pluripotency of derived iPS clones. RESULTS: Ectopic expression of OCT4,SOX2,KLF4,and c-MYC in HPCs and PBMCs resulted in rapid iPSC derivation. Long-term time-lapse imaging revealed efficient iPSC growth under serum- and feeder-free conditions with frequent mitotic events. HPC- and PBMC-derived iPS cells expressed pluripotency-associated markers,including SSEA-4,TRA-1-60,and NANOG. The global gene-expression profiles demonstrated the induction of endogenous pluripotent genes,such as LIN28,TERT,DPPA4,and PODXL,in derived iPSCs. iPSC clones from blood and other cell sources showed similar ultrastructural morphologies and genome-wide gene-expression profiles. On spontaneous and guided differentiation,HPC- and PBMC-derived iPSCs were differentiated into cells of three germ layers,including insulin-producing cells through endodermal lineage,verifying the pluripotency of the blood-derived iPSC clones. CONCLUSIONS: Because the use of blood cells allows minimally invasive tissue procurement under GMP conditions and rapid cellular reprogramming,mobilized HPCs and unmobilized PBMCs would be ideal somatic cell sources for clinical-grade iPSC derivation,especially from diabetes patients complicated by slow-healing wounds. View Publication

We have previously demonstrated that the Src family kinase Yes,the Yes-associated protein (YAP) and TEA domain TEAD2 transcription factor pathway are activated by leukemia inhibitory factor (LIF) and contribute to mouse embryonic stem (mES) cell maintenance of pluripotency and self-renewal. In addition,we have shown that fetal bovine serum (FBS) induces Yes auto-phosphorylation and activation. In the present study we confirm that serum also activates TEAD-dependent transcription in a time- and dose-dependent manner and we identify Inter-α-inhibitor (IαI) as a component in serum capable of activating the Yes/YAP/TEAD pathway by inducing Yes auto-phosphorylation,YAP nuclear localization and TEAD-dependent transcription. The cleaved heavy chain 2 (HC2) sub-component of IαI,is demonstrated to be responsible for this effect. Moreover,IαI is also shown to efficiently increase expression of TEAD-downstream target genes including well-known stem cell factors Nanog and Oct 3/4. IαI is not produced by the ES cells per se but is added to the cells via the cell culture medium containing serum or serum-derived components such as bovine serum albumin (BSA). In conclusion,we describe a novel function of IαI in activating key pluripotency pathways associated with ES cell maintenance and self-renewal. View Publication

BACKGROUND Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage,differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally,current differentiation strategies are hampered by inefficiency,lack of reproducibility,and use of animal-derived products. METHODS To direct the differentiation of hESCs to endothelial subtypes,H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3),basic fibroblast growth factor (bFGF),bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation,the endothelial progenitor cells (CD34(+)CD31(+) cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore,the safety and functionality of these cells upon in vivo transplantation were characterized. RESULTS Sequential modulation of hESCs with GSK-3 inhibitor,bFGF,BMP4 and VEGF resulted in stages reminiscent of primitive streak,early mesoderm/lateral plate mesoderm,and endothelial progenitors under feeder- and serum-free conditions. Furthermore,these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically,the endothelial progenitors differentiated to venous ECs in the absence of VEGF,and to arterial phenotype under low concentrations of VEGF. Additionally,these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore,these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse. CONCLUSIONS We report a simple,rapid,and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery,disease modeling and regenerative medicine in the future. View Publication

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

AIMS Contradictory outcomes from recent clinical trials investigating the transplantation of autologous bone marrow mononuclear cell (BM-MNC) fraction containing stem/progenitor cells to damaged myocardium,following acute myocardial infarction,may be,in part,due to the different cell isolation protocols used. We compared total BM-MNC numbers and its cellular subsets obtained following isolation using Ficoll-Paque and Lymphoprep - two different density gradient media used in the clinical trials. MATERIALS & METHODS Bone marrow samples were taken from patients entered into the REGENERATE-IHD clinical trial after 5 days of subcutaneous granulocyte colony-stimulating factor injections. Each sample was divided equally for BM-MNC isolation using Ficoll-Paque and Lymphoprep,keeping all other procedural steps constant. Isolated fractions were characterized for hematopoietic stem cells,endothelial progenitor cells,T lymphocytes,B lymphocytes and NK cells using cell surface markers CD34(+),CD133(+)VEGFR2(+),CD45(+)CD3(+),CD45(+)CD19(+) and CD45(+)CD16(+)CD56(+),respectively. There were no significant differences in the absolute numbers and percentage cell recovery of various mononuclear cell types recovered following separation using either density gradient media. Cell viability and the proportion of various cell phenotypes investigated were similar between the two media. They were also equally efficient in excluding unwanted red blood cells,granulocytes and platelets from the final cell products. CONCLUSION We demonstrated that the composition and quantity of cell types found within therapeutic BM-MNC preparations for use in clinical trials of cardiac stem cell transplantation are not influenced by the type of density gradient media used when comparing Ficoll-Paque and Lymphoprep. View Publication