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

Tumor-initiating cells (TICs) are defined by their ability to form tumors after xenotransplantation in immunodeficient mice and appear to be relatively rare in most human cancers. Recent data in melanoma indicate that the frequency of TICs increases dramatically via more permissive xenotransplantation conditions,raising the possibility that the true frequency of TICs has been greatly underestimated in most human tumors. We compared the growth of human pancreatic,non-small cell lung,and head and neck carcinomas in NOD/SCID and NSG mice. Although TIC frequency was detected up to 10-fold higher in NSG mice,it remained low (textless1 in 2500 cells) in all cases. Moreover,aldehyde dehydrogenase-positive (ALDH(+)) and CD44(+)CD24(+) cells,phenotypically distinct cells enriched in TICs,were equally tumorigenic in NOD/SCID and NSG mice. Our findings demonstrate that TICs are rare in these cancers and that the identification of TICs and their frequency in other human malignancies should be validated via primary tumors and highly permissive xenotransplantation conditions. View Publication

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct,in the absence of proliferation and multipotent progenitor generation,or indirect,by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state,induced by brief exposure to reprogramming factors,followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells,including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies. View Publication

The tumorigenicity of human pluripotent stem cells (hPSCs) is widely acknowledged as a major obstacle that withholds their application in regenerative medicine. This protocol describes two efficient and robust ways to chemically eliminate the tumor-initiating hPSCs from monolayer culture. The protocol details how to maintain and differentiate hPSCs,how to apply chemical inhibitors to cultures of hPSCs and their differentiated progeny,and how to assess the purity of the resultant cell cultures using in vitro and in vivo assays. It also describes how to rescue the cytotoxic effect. The elimination and the rescue assay can be completed within 3-5 d,the in vitro assessment requires another day,and the in vivo assessment requires up to 12 additional weeks. View Publication

Human pluripotent stem cells (HPSCs) cultured in conditions that maintain pluripotency via FGF and TGFβ signaling have been described as being in a primed state. These cells have been shown to exhibit characteristics more closely related to mouse epiblast-derived stem cells than to so called naïve mouse PSCs said to possess a more ground state pluripotency that mimics the early mouse embryo inner cell mass. Initial attempts to create culture conditions favorable for generation of naïve HPSCs from primed HPSCs has required the use of mouse embryonic fibroblasts as a feeder layer to support this transition. A protocol for the routine derivation and maintenance of naïve HPSCs in completely defined conditions is highly desirable for stem cell researchers to enhance the study and clinical translation of naïve HPSCs. Here we describe a standard protocol for transitioning primed HPSCs to a naïve state using commercial RSet media and xeno-free recombinant vitronectin. View Publication

Recombinant human GM-CSF has been expressed as a fusion protein in E. coli in the form of inclusion bodies. Using denaturing agents,acid cleavage and sulfitolysis,the biologically inactive GM-CSF protein could be highly purified and additionally renaturated under suitable reoxidizing conditions. The thorough repair of the two disulfide bridges could be confirmed by sequencing fragments obtained by tryptic digestion. Refolding of the molecule has been studied by CD spectrometry and identity by Western blotting and SDS-PAGE analysis. As could be demonstrated,full biological activity (colony-forming assay with fresh human bone marrow cells) was restored during renaturation of the GM-CSF protein. Further proof of biological equivalence of the E. coli-derived protein with a yeast-derived biologically active rh GM-CSF has been published elsewhere. View Publication

Human-induced airway basal cells (hiBCs) derived from human-induced pluripotent stem cells (hiPSCs) offer a promising cell model for studying lung diseases,regenerative medicine,and developing new gene therapy methods. We analyzed existing differentiation protocols and proposed our own protocol for obtaining hiBCs,which involves step-by-step differentiation of hiPSCs into definitive endoderm,anterior foregut endoderm,NKX2.1+ lung progenitors,and cultivation on basal cell medium with subsequent cell sorting using the surface marker CD271 (NGFR). We derived hiBCs from two healthy cell lines and three cell lines with cystic fibrosis (CF). The obtained hiBCs,expressing basal cell markers (NGFR,KRT5,and TP63),could differentiate into lung organoids (LOs). We demonstrated that LOs derived from hiBCs can assess cystic fibrosis transmembrane conductance regulator (CFTR) channel function using the forskolin-induced swelling (FIS) assay. We also carried out non-viral (electroporation) and viral (recombinant adeno-associated virus (rAAV)) serotypes 6 and 9 and recombinant adenovirus (rAdV) serotype 5 transgene delivery to hiBCs and showed that rAAV serotype 6 is most effective against hiBCs,potentially applicable for gene therapy research. View Publication

Recent population studies provide clues that the use of metformin may be associated with reduced incidence and improved prognosis of certain cancers. This drug is widely used in the treatment of type 2 diabetes,where it is often referred to as an insulin sensitizer" because it not only lowers blood glucose but also reduces the hyperinsulinemia associated with insulin resistance. As insulin and insulin-like growth factors stimulate proliferation of many normal and transformed cell types� View Publication