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

The recently developed ability to differentiate primary adult stem cells and induced pluripotent stem cells (iPSCs) into cardiomyocytes is providing unprecedented opportunities to produce an unlimited supply of cardiomyocytes for use in patients with heart disease. Here,we examine the evidence for the preclinical use of such cells for successful heart regeneration. We also describe advances in the identification of new cardiac molecular and cellular targets to induce proliferation of cardiomyocytes for heart regeneration. Such new advances are paving the way for a new innovative drug development process for the treatment of heart disease. View Publication

TOX is a DNA-binding factor required for development of CD4(+) T cells,natural killer T cells and regulatory T cells. Here we document that both natural killer (NK) cell development and lymphoid tissue organogenesis were also inhibited in the absence of TOX. We found that the development of lymphoid tissue-inducer cells,a rare subset of specialized cells that has an integral role in lymphoid tissue organogenesis,required TOX. Tox was upregulated considerably in immature NK cells in the bone marrow,consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus,many cell lineages of the immune system share a TOX-dependent step for development. View Publication

Fibroblast growth factor receptor 1 (FGFR1) is overexpressed in multiple types of solid tumors,including head and neck squamous cell carcinoma (HNSCC). Being associated with poor prognosis,FGFR1 is a potential therapeutic target for aggressive tumors. T cell-based cancer immunotherapy has played a central role in novel cancer treatments. However,the potential of antitumor immunotherapy targeting FGFR1 has not been investigated. Here,we showed that FGFR-tyrosine kinase inhibitors (TKIs) augmented antitumor effects of immune checkpoint inhibitors in an HNSCC mouse model and upregulated tumoral MHC class I and MHC class II expression in vivo and in vitro. This upregulation was associated with the mitogen-activated protein kinase signaling pathway,which is a crucial pathway for cancer development through FGFR signaling. Moreover,we identified an FGFR1-derived peptide epitope (FGFR1305-319) that could elicit antigen-reactive and multiple HLA-restricted CD4+ T cell responses. These T cells showed direct cytotoxicity against tumor cells that expressed FGFR1. Notably,FGFR-TKIs augmented antitumor effects of FGFR1-reactive T cells against human HNSCC cells. These results indicate that the combination of FGFR-TKIs with immunotherapy,such as an FGFR1-targeting peptide vaccine or immune checkpoint inhibitor,could be a novel and robust immunologic approach for treating patients with FGFR1-expressing cancer cells. View Publication

The human peripheral B-cell compartment displays a large population of immunoglobulin M-positive,immunoglobulin D-positive CD27(+) (IgM(+)IgD(+)CD27(+)) memory" B cells carrying a mutated immunoglobulin receptor. By means of phenotypic analysis� View Publication

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34(+) cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sac-derived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I (zeta(2)epsilon(2)) and the orthochromatic erythroblasts hemoglobin Gower II (alpha(2)epsilon(2)). This suggests that the switch from Hb Gower I to Hb Gower II,the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34(+) cells that differentiate into more developmentally mature,fetal liver-like erythroblasts,that are smaller,express mostly fetal hemoglobin,and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated,and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary,because the most mature cells that can be produced with current systems express less than 2% adult beta-globin mRNA. View Publication

The purpose of this study was to examine the in vitro and in vivo osteogenic potential of human induced pluripotent stem cells (hiPSCs) against that of human bone marrow mesenchymal stem cells (hBMMSCs). Embryoid bodies (EBs),which were formed from undifferentiated hiPSCs,were dissociated into single cells and underwent osteogenic differentiation using the same medium as hBMMSCs for 14 days. Osteoinduced hiPSCs were implanted on the critical-size calvarial defects and long bone segmental defects in rats. The healing of defects was evaluated after 8 weeks and 12 weeks of implantation,respectively. Osteoinduced hiPSCs showed relatively lower and delayed in vitro expressions of the osteogenic marker COL1A1 and bone sialoprotein,as well as a weaker osteogenic differentiation through alkaline phosphatase staining and mineralization through Alizarin red staining compared with hBMMSCs. Calvarial defects treated with osteoinduced hiPSCs had comparable quality of new bone formation,including full restoration of bone width and robust formation of trabeculae,to those treated with hBMMSCs. Both osteoinduced hiPSCs and hBMMSCs persisted in regenerated bone after 8 weeks of implantation. In critical-size long bone segmental defects,osteoinduced hiPSC treatment also led to healing of segmental defects comparable to osteoinduced hBMMSC treatment after 12 weeks. In conclusion,despite delayed in vitro osteogenesis,hiPSCs have an in vivo osteogenic potential as good as hBMMSCs. View Publication

The clinical use of human embryonic stem cells (hESCs) requires efficient cellular expansion that must be paired with an ability to generate specialized progeny through differentiation. Self-renewal and differentiation are deemed inherent hallmarks of hESCs and a growing body of evidence suggests that initial culture conditions dictate these two aspects of hESC behavior. Here,we reveal that defined culture conditions using commercial mTeSR1 media augment the expansion of hESCs and enhance their capacity for neural differentiation at the expense of hematopoietic lineage competency without affecting pluripotency. This culture-induced modification was shown to be reversible,as culture in mouse embryonic fibroblast-conditioned media (MEF-CM) in subsequent passages allowed mTeSR1-expanded hESCs to re-establish hematopoietic differentiation potential. Optimal yield of hematopoietic cells can be achieved by expansion in mTeSR1 followed by a recovery period in MEF-CM. Furthermore,the lineage propensity to hematopoietic and neural cell types could be predicted via analysis of surrogate markers expressed by hESCs cultured in mTeSR1 versus MEF-CM,thereby circumventing laborious in vitro differentiation assays. Our study reveals that hESCs exist in a range of functional states and balance expansion with differentiation potential,which can be modulated by culture conditions in a predictive and quantitative manner. Stem Cells 2015;33:1142-1152. View Publication

Human embryonic stem cells (hESCs) are considered a potential alternative to cadaveric islets as a source of transplantable cells for treating patients with diabetes. We previously described a differentiation protocol to generate pancreatic progenitor cells from hESCs,composed of mainly pancreatic endoderm (PDX1/NKX6.1-positive),endocrine precursors (NKX2.2/synaptophysin-positive,hormone/NKX6.1-negative),and polyhormonal cells (insulin/glucagon-positive,NKX6.1-negative). However,the relative contributions of NKX6.1-negative versus NKX6.1-positive cell fractions to the maturation of functional β-cells remained unclear. To address this question,we generated two distinct pancreatic progenitor cell populations using modified differentiation protocols. Prior to transplant,both populations contained a high proportion of PDX1-expressing cells (˜85%-90%) but were distinguished by their relatively high (˜80%) or low (˜25%) expression of NKX6.1. NKX6.1-high and NKX6.1-low progenitor populations were transplanted subcutaneously within macroencapsulation devices into diabetic mice. Mice transplanted with NKX6.1-low cells remained hyperglycemic throughout the 5-month post-transplant period whereas diabetes was reversed in NKX6.1-high recipients within 3 months. Fasting human C-peptide levels were similar between groups throughout the study,but only NKX6.1-high grafts displayed robust meal-,glucose- and arginine-responsive insulin secretion as early as 3 months post-transplant. NKX6.1-low recipients displayed elevated fasting glucagon levels. Theracyte devices from both groups contained almost exclusively pancreatic endocrine tissue,but NKX6.1-high grafts contained a greater proportion of insulin-positive and somatostatin-positive cells,whereas NKX6.1-low grafts contained mainly glucagon-expressing cells. Insulin-positive cells in NKX6.1-high,but not NKX6.1-low grafts expressed nuclear MAFA. Collectively,this study demonstrates that a pancreatic endoderm-enriched population can mature into highly functional β-cells with only a minor contribution from the endocrine subpopulation. View Publication

The major goal of researchers and oncologists is to develop promising ground for novel therapeutic strategies to prevent recurrence or relapse of cancer. Recent evidences suggest that a subset of cells called cancer stem cells (CSCs) are present within the tumor mass which possess tumorigenic capacity and may be responsible for propagation,relapse,and metastatic dissemination. These cells have certain stem cell-like properties,e.g. quiescence,selfrenewal,asymmetric division,and multidrug resistance which allow them to drive tumor growth and evade conventional therapies. A number of markers and assays have been designed to isolate and characterize the CSC population from the bulk tumor. The objective now is to selectively target the CSCs in order to eliminate the tumor from root,overcoming the emergence of clones capable of evading traditional therapy. This approach may help in increasing the overall disease-free survival in some cancers. View Publication

Genetic manipulation of human embryonic stem cells (hESC) has been limited by their general resistance to common methods used to introduce exogenous DNA or RNA. Efficient and high throughput transfection of nucleic acids into hESC would be a valuable experimental tool to manipulate these cells for research and clinical applications. We investigated the ability of two commercially available electroporation systems,the Nucleofection® 96-well Shuttle® System from Lonza and the Neon™ Transfection System from Invitrogen to efficiently transfect hESC. Transfection efficiency was measured by flow cytometry for the expression of the green fluorescent protein and the viability of the transfected cells was determined by an ATP catalyzed luciferase reaction. The transfected cells were also analyzed by flow cytometry for common markers of pluripotency. Both systems are capable of transfecting hESC at high efficiencies with little loss of cell viability. However,the reproducibility and the ease of scaling for high throughput applications led us to perform more comprehensive tests on the Nucleofection® 96-well Shuttle® System. We demonstrate that this method yields a large fraction of transiently transfected cells with minimal loss of cell viability and pluripotency,producing protein expression from plasmid vectors in several different hESC lines. The method scales to a 96-well plate with similar transfection efficiencies at the start and end of the plate. We also investigated the efficiency with which stable transfectants can be generated and recovered under antibiotic selection. Finally,we found that this method is effective in the delivery of short synthetic RNA oligonucleotides (siRNA) into hESC for knockdown of translation activity via RNA interference. Our results indicate that these electroporation methods provide a reliable,efficient,and high-throughput approach to the genetic manipulation of hESC. View Publication

Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily,although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged,and are associated with elevated transcription of HERVH,a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors,including LBP9,recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts,including pluripotency-modulating long non-coding RNAs. Disruption of LBP9,HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs,and establish novel primate-specific transcriptional circuitry regulating pluripotency. View Publication

Combinatorial libraries were screened for molecules that induce mouse myogenic lineage committed cells to dedifferentiate in vitro. A 2,6-disubstituted purine,reversine,was discovered that induces lineage reversal of C2C12 cells to become multipotent progenitor cells which can redifferentiate into osteoblasts and adipocytes. This and other such molecules are likely to provide new insights into the molecular mechanisms that control cellular dedifferentiation and may ultimately be useful to in vivo stem cell biology and therapy. View Publication