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

We have used in vitro and mouse xenograft models to examine the interaction between breast cancer stem cells (CSC) and bone marrow-derived mesenchymal stem cells (MSC). We show that both of these cell populations are organized in a cellular hierarchy in which primitive aldehyde dehydrogenase expressing mesenchymal cells regulate breast CSCs through cytokine loops involving IL6 and CXCL7. In NOD/SCID mice,labeled MSCs introduced into the tibia traffic to sites of growing breast tumor xenografts where they accelerated tumor growth by increasing the breast CSC population. With immunochemistry,we identified MSC-CSC niches in these tumor xenografts as well as in frozen sections from primary human breast cancers. Bone marrow-derived MSCs may accelerate human breast tumor growth by generating cytokine networks that regulate the CSC population. View Publication

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells,when isolated and cultured in vitro,were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition,Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor. View Publication

In this study,we sought to establish a novel method to prospectively and dynamically identify live human oligodendrocyte precursor cells (OPCs) and oligodendrocyte lineage cells from brain dissociates and pluripotent stem cell culture. We selected a highly conserved enhancer element of the Sox10 gene,known as MCS5,which directs reporter expression to oligodendrocyte lineage cells in mouse and zebrafish. We demonstrate that lentiviral Sox10-MCS5 induced expression of GFP at high levels in a subpopulation of human CD140a/PDGF??R-sorted OPCs as well as their immature oligodendrocyte progeny. Furthermore,we show that almost all Sox10-MCS5:GFPhigh cells expressed OPC antigen CD140a and human OPCs expressing SOX10,OLIG2,and PDGFRA mRNAs could be prospectively identified using GFP based fluorescence activated cells sorting alone. Additionally,we established a human induced pluripotent cell (iPSC) line transduced with the Sox10-MCS5:GFP reporter using a Rex-Neo cassette. Similar to human primary cells,GFP expression was restricted to embryoid bodies containing both oligodendrocyte progenitor and oligodendrocyte cells and co-localized with NG2 and O4-positive cells respectively. As such,we have developed a novel reporter system that can track oligodendrocyte commitment in human cells,establishing a valuable tool to improve our understanding and efficiency of human oligodendrocyte derivation. ?? 2013 Elsevier Inc. View Publication

The developmental pathway for human megakaryocytes remains unclear and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis,we have identified a cluster of cells within immature hematopoietic stem and progenitor cell populations that specifically express genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34(+)CD38(+)IL-3Rα(dim)CD45RA(-) common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte/macrophage potential,but exhibited robust differentiation into the megakaryocyte lineage at a high frequency,both in vivo and in vitro The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly,the CD41(+) CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis,independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia,the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus,the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis. View Publication

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However,many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First,we developed functional re-coded TALEs (reTALEs),which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design,we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks. View Publication

Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast,induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs,which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology,immunophenotype,in vitro differentiation potential,and gene expression profiles as primary MSCs. However,iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics,whereas tissue-specific,senescence-associated,and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion,but they remained rejuvenated with regard to age-related DNAm. Overall,iPS-MSCs are similar to MSCs,but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns - particularly of DNAm patterns associated with tissue type and aging. View Publication

Normal prion protein (PrP(c)),an essential substrate for development of prion disease,is widely distributed in hematopoietic cells. Recent evidence that variant Creutzfeldt-Jakob disease can be transmitted by transfusion of red cell preparations has highlighted the need for a greater understanding of the biology of PrP(c) in blood and blood-forming tissues. Here,we show that in contrast to another glycosylphosphoinositol-anchored protein CD59,PrP(c) at the cell surface of cultured human erythroblasts is rapidly internalized through the endosomal pathway,where it colocalizes with the tetraspanin CD63. In the plasma membrane,PrP(c) colocalizes with the tetraspanin CD81. Cross-linking with anti-PrP(c) or anti-CD81 causes clustering of PrP(c) and CD81,suggesting they can share the same microdomain. These data are consistent with a role for tetraspanin-enriched microdomains in trafficking of PrP(c). These results,when taken together with recent evidence that exosomes released from cells as a result of endosomal-mediated recycling to the plasma membrane contain prion infectivity,provide a pathway for the propagation of prion diseases. View Publication

Induced pluripotent stem (iPS) cells hold the potential to revolutionize regenerative medicine through their capacity to generate cells of diverse lineages for future patient-specific cell-based therapies. To facilitate the transition of iPS cells to clinical practice,a variety of technologies have been developed for transgene-free pluripotency reprogramming. We recently reported efficient iPS cell generation from human fibroblasts using synthetic modified mRNAs. Here we describe a stepwise protocol for the generation of modified mRNA-derived iPS cells from primary human fibroblasts,focusing on the critical parameters including medium choice,quality control,and optimization steps needed for synthesizing modified mRNAs encoding reprogramming factors and introducing these into cells over the course of 2-3 weeks to ensure successful reprogramming. The protocol described herein is for reprogramming of human fibroblasts to pluripotency; however,the properties of modified mRNA make it a powerful platform for protein expression,which has broad applicability in directed differentiation,cell fate specification and therapeutic applications. View Publication

Cardiomyocytes (CM) derived from human embryonic stem cells (hESC) are used for cardio-toxicity evaluation and tested in many preclinical trials for their potential use in regenerative therapeutics. As more efficient CM differentiation protocols are developed,reliable automated platforms for characterization and detection are needed. An automated time-resolved video analysis and management system (TVAMS) has been developed for the evaluation of hESC differentiation to CM. The system was used for monitoring the kinetics of embryoid bodies (EB) generation (numbers and size) and differentiation into beating EBs (percentage beating area and beating EB count) in two differentiation protocols. We show that the percentage beating areas of EBs (from total area of the EBs) is a more sensitive and better predictor of CM differentiation efficiency than percentage of beating EBs (from total EBs) as the percentage beating areas of EBs correlates with cardiac troponin-T and myosin heavy chain expression levels. TVAMS can also be used to evaluate the effect of drugs and inhibitors (e.g. isoproterenol and ZD7288) on CM beating frequency. TVAMS can reliably replace the commonly practiced,time consuming,manual counting of total and beating EBs during CM differentiation. TVAMS is a high-throughput non-invasive video imaging platform that can be applied for the development of new CM differentiation protocols,as well as a tool to conduct CM toxicology assays. View Publication

INTRODUCTION: The physiological signals that direct the division and differentiation of the zygote to form a blastocyst,and subsequent embryonic stem cell division and differentiation during early embryogenesis,are unknown. Although a number of growth factors,including the pregnancy-associated hormone human chorionic gonadotropin (hCG) are secreted by trophoblasts that lie adjacent to the embryoblast in the blastocyst,it is not known whether these growth factors directly signal human embryonic stem cells (hESCs).backslashnbackslashnMETHODS: Here we used hESCs as a model of inner cell mass differentiation to examine the hormonal requirements for the formation of embryoid bodies (EB's; akin to blastulation) and neuroectodermal rosettes (akin to neurulation).backslashnbackslashnRESULTS: We found that hCG promotes the division of hESCs and their differentiation into EB's and neuroectodermal rosettes. Inhibition of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) signaling suppresses hESC proliferation,an effect that is reversed by treatment with hCG. hCG treatment rapidly upregulates steroidogenic acute regulatory protein (StAR)-mediated cholesterol transport and the synthesis of progesterone (P4). hESCs express P4 receptor A,and treatment of hESC colonies with P4 induces neurulation,as demonstrated by the expression of nestin and the formation of columnar neuroectodermal cells that organize into neural tubelike rosettes. Suppression of P4 signaling by withdrawing P4 or treating with the P4-receptor antagonist RU-486 inhibits the differentiation of hESC colonies into EB's and rosettes.backslashnbackslashnCONCLUSIONS: Our findings indicate that hCG signaling via LHCGR on hESC promotes proliferation and differentiation during blastulation and neurulation. These findings suggest that trophoblastic hCG secretion and signaling to the adjacent embryoblast could be the commencement of trophic support by placental tissues in the growth and development of the human embryo. View Publication