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

The reliable derivation of induced pluripotent stem cells (iPSCs) from a noninvasive autologous source at birth would facilitate the study of patient-specific in vitro modeling of congenital diseases and would enhance ongoing efforts aimed at developing novel cell-based treatments for a wide array of fetal and pediatric disorders. Accordingly,we have successfully generated iPSCs from human fetal chorionic somatic cells extracted from term pregnancies by ectopic expression of OCT4,SOX2,KLF4,and cMYC. The isolated parental somatic cells exhibited an immunophenotypic profile consistent with that of chorionic mesenchymal stromal cells (CMSCs). CMSC-iPSCs maintained pluripotency in feeder-free systems for more than 15 passages based on morphology,immunocytochemistry,and gene expression studies and were capable of embryoid body formation with spontaneous trilineage differentiation. CMSC-iPSCs could be selectively differentiated in vitro into various germ layer derivatives,including neural stem cells,beating cardiomyocytes,and definitive endoderm. This study demonstrates the feasibility of term placental chorion as a novel noninvasive alternative to dermal fibroblasts and cord blood for human perinatal iPSC derivation and may provide additional insights regarding the reprogramming capabilities of extra-embryonic tissues as they relate to developmental ontogeny and perinatal tissue engineering applications. View Publication

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1,ALDH3A1,and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity,and that this activity is associated with ALDH1A1 expression. Isolated ALDH(+) lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared with their ALDH(-) counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH(+) cells. Suppression of the Notch pathway by treatment with either a γ-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH(+) lung cancer cells,commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together,these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential,that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis,and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH(+) component,implicating Notch signaling in lung cancer stem cell maintenance. View Publication

The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways,the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK,i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose,heat shock),AICAR does not perturb the cellular contents of ATP,ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation,causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase),and almost total cessation of two of the known target pathways,i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase,previously demonstrated in cell-free assays,also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade. View Publication

Human progenitors of the megakaryocyte (Mk) lineage were detected by their ability to generate colonies-containing from 3 to textgreater 100 Mk,detectable as glycoprotein IIb/IIIa+ cells in APAAP-stained whole mount agarose cultures. Optimal growth conditions were achieved through the use of a defined serum substitute and a suitable cocktail of recombinant cytokines. Under these culture conditions,the smallest Mk-containing colonies (CFC-Mk) were detectable within a week followed by colonies containing larger numbers of Mk over the ensuing 2 weeks. The total number of CFC-Mk at 18-21 d was linearly related to the number of cells plated. Variation in the cytokines added showed that thrombopoietin (TPO) or IL-3 alone would support the formation of large numbers of CFC-Mk. However,optimal yields of colonies containing cells of both Mk and non-Mk lineages required the addition of other growth factors,of which a combination of IL-3,IL-6,GM-CSF and Steel factor (SF) +/- TPO was the best of those tested. The further addition of erythropoietin to this combination reduced the number of large pure' Mk colonies seen and in their place a corresponding number of mixed erythroid-Mk colonies became detectable. Flt3-ligand alone was unable to support the growth of CFC-Mk nor did it enhance their growth when combined with other factors. Plating of FACS-sorted sub-populations of CD34+ marrow cells in both serum-free agarose and methylcellulose assays demonstrated that most CFC-Mk are generated from CD34+ cells that are CD45RA- and CD71+� View Publication

Activation of ErbB4 receptor signaling is instrumental in heart development,lack of which results in embryonic lethality. However,mechanism governing its intracellular signaling remains elusive. Using human pluripotent stem cells,we show that ErbB4 is critical for cardiogenesis whereby its genetic knockdown results in loss of cardiomyocytes. Phospho-proteome profiling and Western blot studies attribute this loss to inactivation of p38$\$ isoform which physically interacts with NKx2.5 and GATA4 transcription factors. Post-cardiomyocyte formation p38$\$/NKx2.5 downregulation is followed by p38$\$/MEF2c upregulation suggesting stage-specific developmental roles of p38 MAPK isoforms. Knockdown of p38$\$ similarly disrupts cardiomyocyte formation in spite of the presence of NKx2.5. Cell fractionation and NKx2.5 phosphorylation studies suggest inhibition of ErbB4-p38$\$ hinders NKx2.5 nuclear translocation during early cardiogenesis. This study reveals a novel pathway that directly links ErbB4 and p38$\$ the transcriptional machinery of NKx2.5-GATA4 complex which is critical for cardiomyocyte formation during mammalian heart development. View Publication

Lupus glomerulonephritis (GN) is an autoimmune disease characterized by immune complex-deposition,complement activation and glomerular inflammation. In lupus-prone NZM2328 mice,the occurrence of lupus GN was accompanied by a decrease in Treg cells and an increase in proinflammatory cytokine-producing T cells. Because IL-33 in addition to IL-2 has been shown to be important for Treg cell proliferation and ST2 (IL-33 receptor) positive Treg cells are more potent in suppressor activity,a hybrid cytokine with active domains of IL-2 and IL-33 was generated to target the ST2+ Treg cells as a therapeutic agent to treat lupus GN. Three mouse models were used: spontaneous and Ad-IFNalpha- accelerated lupus GN in NZM2328 and the lymphoproliferative autoimmune GN in MRL/lpr mice. Daily injections of IL233 for 5 days prevented Ad-IFNalpha-induced lupus GN and induced remission of spontaneous lupus GN. The remission was permanent in that no relapses were detected. The remission was accompanied by persistent elevation of Treg cells in the renal lymph nodes. IL233 is more potent than IL-2 and IL-33 either singly or in combination in the treatment of lupus GN. The results of this study support the thesis that IL233 should be considered as a novel agent for treating lupus GN. View Publication

Epithelial-mesenchymal transition (EMT) plays important roles in various physiological and pathological processes,and is regulated by signaling pathways mediated by cytokines,including transforming growth factor beta (TGFbeta). Embryonic endothelial cells also undergo differentiation into mesenchymal cells during heart valve formation and aortic maturation. However,the molecular mechanisms that regulate such endothelial-mesenchymal transition (EndMT) remain to be elucidated. Here we show that TGFbeta plays important roles during mural differentiation of mouse embryonic stem cell-derived endothelial cells (MESECs). TGFbeta2 induced the differentiation of MESECs into mural cells,with a decrease in the expression of the endothelial marker claudin 5,and an increase in expression of the mural markers smooth muscle alpha-actin,SM22alpha and calponin,whereas a TGFbeta type I receptor kinase inhibitor inhibited EndMT. Among the transcription factors involved in EMT,Snail was induced by TGFbeta2 in MESECs. Tetracycline-regulated expression of Snail induced the differentiation of MESECs into mural cells,whereas knockdown of Snail expression abrogated TGFbeta2-induced mural differentiation of MESECs. These results indicate that Snail mediates the actions of endogenous TGFbeta signals that induce EndMT. View Publication

To investigate whether aldehyde dehydrogenase-1 (ALDH-1) in human lung cancer can be used as a sorting marker for stem cells in targeted therapies against human lung cancer. Spheres were induced by incubating cancer cells in a serum-free medium and formed with epidermal growth factor and fibroblast growth factor-10 (FGF10). Spheroid cells were combined with flow cytometry using the Aldefluor reagent to separate the SSCloALDEbr (ALDH-1-positive) cells. Cancer stem cells (CSCs) were characterized by their proliferation,colony formation,and tumorigenesis in nude mice and using phenotypic analysis. Float-growing spheres (pulmospheres") were developed after SPC-A1 cells were cultured in a serum-free medium. The resultant sphere-forming cells included ALDH-1-positive cells as high as 15.13%. ALDH-1-positive CSCs have high proliferative ability� View Publication

Haematopoietic stem cell (HSC) gene therapy has demonstrated potential to treat many diseases. However,current state of the art requires sophisticated ex vivo gene transfer in a dedicated Good Manufacturing Practices facility,limiting availability. An automated process would improve the availability and standardized manufacture of HSC gene therapy. Here,we develop a novel program for semi-automated cell isolation and culture equipment to permit complete benchtop generation of gene-modified CD34+ blood cell products for transplantation. These cell products meet current manufacturing quality standards for both mobilized leukapheresis and bone marrow,and reconstitute human haematopoiesis in immunocompromised mice. Importantly,nonhuman primate autologous gene-modified CD34+ cell products are capable of stable,polyclonal multilineage reconstitution with follow-up of more than 1 year. These data demonstrate proof of concept for point-of-care delivery of HSC gene therapy. Given the many target diseases for gene therapy,there is enormous potential for this approach to treat patients on a global scale. View Publication

Pluripotent human embryonic stem cells (hESCs) are a potential renewable cell source for regenerative medicine and drug testing. To obtain adequate cell numbers for these applications,there is a need to develop scalable cell culture platforms to propagate hESCs. In this study,we encapsulated hESCs in calcium alginate microfibers as single cells,for expansion and differentiation under chemically defined conditions. hESCs were suspended in 1% (w/v) alginate solution at high cell density (textgreater10(7) cells/mL) and extruded at 5 m/min into a low calcium concentration bath (10 mM) for gelation. Mild citrate buffer (2.5 mM),which did not affect hESCs viability,was used to release the cells from the calcium alginate hydrogel. Encapsulation as single cells was critical,as this allowed the hESCs to grow in the form of relatively small and uniform aggregates. This alginate microfiber system allowed for expansion of an hESC line,HUES7,for up to five passages while maintaining pluripotency. Immunohistochemistry,polymerase chain reaction,and other analyses showed that passage 5 (P5) HUES7 cells expressed proteins and genes characteristic of pluripotent stem cells,possessed normal karyotype,and were able to form representative tissues of the three embryonic germ layers in vitro and in vivo. Encapsulated HUES7 cells at P5 could also be induced to directly differentiate into liver-like cells. Collectively,our experiments show that the alginate microfiber system can be used as a three-dimensional cell culture platform for long-term expansion and differentiation of hESCs under defined conditions. View Publication