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

The future of safe cell-based therapy rests on overcoming teratoma/tumor formation,in particular when using human pluripotent stem cells (hPSCs),such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation,complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile,we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e.,survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors,leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular,a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g.,quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast,differentiated cell types (e.g.,dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together,these results provide the proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy." View Publication

Imatinib is currently the first line therapy for newly diagnosed patients with chronic myeloid leukemia. However,20-25% of patients do not achieve durable complete cytogenetic responses. The mechanism underlying this primary resistance is unknown,but variations in BCR-ABL1 kinase activity may play a role and can be investigated by measuring the autophosphorylation levels of BCR-ABL1 or of a surrogate target such as Crkl. In this study we used flow cytometry to investigate the in vitro inhibition of Crkl phosphorylation by imatinib in CD34(+) cells in diagnostic samples from two groups of patients distinguished by their cytogenetic response. No difference in inhibition of Crkl phosphorylation was observed in the two groups. The observation that increasing the dose of imatinib in vivo did not increase the level of cytogenetic response in some non-responders suggests that in at least a proportion of patients imatinib resistance may be due to activation of BCR-ABL1-independent pathway. View Publication

The immunodeficiency disorder,X-linked agammaglobulinemia (XLA),results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA,we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice,a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells,treated mice showed significant,albeit incomplete,rescue of mature B cells in the bone marrow,peripheral blood,spleen,and peritoneal cavity,and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression,viral integration,and partial functional responses,consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA. View Publication

Activating mutations in signaling molecules,such as JAK2-V617F,have been associated with myeloproliferative neoplasms (MPNs). Mice lacking the inhibitory adaptor protein Lnk display deregulation of thrombopoietin/thrombopoietin receptor signaling pathways and exhibit similar myeloproliferative characteristics to those found in MPN patients,suggesting a role for Lnk in the molecular pathogenesis of these diseases. Here,we showed that LNK levels are up-regulated and correlate with an increase in the JAK2-V617F mutant allele burden in MPN patients. Using megakaryocytic cells,we demonstrated that Lnk expression is regulated by the TPO-signaling pathway,thus indicating an important negative control loop in these cells. Analysis of platelets derived from MPN patients and megakaryocytic cell lines showed that Lnk can interact with JAK2-WT and V617F through its SH2 domain,but also through an unrevealed JAK2-binding site within its N-terminal region. In addition,the presence of the V617F mutation causes a tighter association with Lnk. Finally,we found that the expression level of the Lnk protein can modulate JAK2-V617F-dependent cell proliferation and that its different domains contribute to the inhibition of multilineage and megakaryocytic progenitor cell growth in vitro. Together,our results indicate that changes in Lnk expression and JAK2-V617F-binding regulate JAK2-mediated signals in MPNs. View Publication

Progressive resistance exercise training (PRT) is the most effective known intervention for combating aging skeletal muscle atrophy. However,the hypertrophic response to PRT is variable,and this may be due to muscle inflammation susceptibility. Metformin reduces inflammation,so we hypothesized that metformin would augment the muscle response to PRT in healthy women and men aged 65 and older. In a randomized,double-blind trial,participants received 1,700 mg/day metformin (N = 46) or placebo (N = 48) throughout the study,and all subjects performed 14 weeks of supervised PRT. Although responses to PRT varied,placebo gained more lean body mass (p = .003) and thigh muscle mass (p {\textless} .001) than metformin. CT scan showed that increases in thigh muscle area (p = .005) and density (p = .020) were greater in placebo versus metformin. There was a trend for blunted strength gains in metformin that did not reach statistical significance. Analyses of vastus lateralis muscle biopsies showed that metformin did not affect fiber hypertrophy,or increases in satellite cell or macrophage abundance with PRT. However,placebo had decreased type I fiber percentage while metformin did not (p = .007). Metformin led to an increase in AMPK signaling,and a trend for blunted increases in mTORC1 signaling in response to PRT. These results underscore the benefits of PRT in older adults,but metformin negatively impacts the hypertrophic response to resistance training in healthy older individuals. ClinicalTrials.gov Identifier: NCT02308228. View Publication

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders,resulting in early-onset,therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy,severe developmental delay,progressive microcephaly,visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent,homozygous variant (chr2:64083454A {\textgreater} G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform,which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells,leading to altered glycogen metabolism,upregulated unfolded protein response and premature neuronal differentiation,as modeled during pluripotent stem cell differentiation in vitro. In contrast,the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly,it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease,even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies. View Publication

The advent of the human-induced pluripotent stem cell (hiPSC) technology has transformed biomedical research,providing new tools for human disease modeling,drug development,and regenerative medicine. To fulfill its unique potential in the cardiovascular field,efficient methods should be developed for high-resolution,large-scale,long-term,and serial functional cellular phenotyping of hiPSC-derived cardiomyocytes (hiPSC-CMs). To achieve this goal,we combined the hiPSC technology with genetically encoded voltage (ArcLight) and calcium (GCaMP5G) fluorescent indicators. Expression of ArcLight and GCaMP5G in hiPSC-CMs permitted to reliably follow changes in transmembrane potential and intracellular calcium levels,respectively. This allowed monitoring short- and long-term changes in action-potential and calcium-handling properties and the development of arrhythmias in response to several pharmaceutical agents and in hiPSC-CMs derived from patients with different inherited arrhythmogenic syndromes. Combining genetically encoded fluorescent reporters with hiPSC-CMs may bring a unique value to the study of inherited disorders,developmental biology,and drug development and testing. View Publication

Current therapies for delayed- or nonunion bone fractures are still largely ineffective. Previous studies indicated that the VEGF homolog placental growth factor (PlGF) has a more significant role in disease than in health. Therefore we investigated the role of PlGF in a model of semi-stabilized bone fracture healing. Fracture repair in mice lacking PlGF was impaired and characterized by a massive accumulation of cartilage in the callus,reminiscent of delayed- or nonunion fractures. PlGF was required for the early recruitment of inflammatory cells and the vascularization of the fracture wound. Interestingly,however,PlGF also played a role in the subsequent stages of the repair process. Indeed in vivo and in vitro findings indicated that PlGF induced the proliferation and osteogenic differentiation of mesenchymal progenitors and stimulated cartilage turnover by particular MMPs. Later in the process,PlGF was required for the remodeling of the newly formed bone by stimulating osteoclast differentiation. As PlGF expression was increased throughout the process of bone repair and all the important cell types involved expressed its receptor VEGFR-1,the present data suggest that PlGF is required for mediating and coordinating the key aspects of fracture repair. Therefore PlGF may potentially offer therapeutic advantages for fracture repair. View Publication

Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin,causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs,when cotransfected with recombinatory donor DNA fragments,can promote single base-pair modification at the start of the second intron of the beta-globin gene,the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose,sequence,cell-cycle stage,and the presence of a homologous donor DNA molecule. Enhanced recombination,with frequencies up to 0.4%,was observed with use of the lysomotropic agent chloroquine. Finally,we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells,including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable,site-specific modification of disease-related genes in human cells. View Publication

Recent evidence suggests that pancreatic cancer and other solid tumors contain a subset of tumorigenic cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. Sorafenib (SO) is a promising new multikinase inhibitor for treatment of advanced kidney and liver cancers. We report here targeting of pancreatic cancer stem cells (CSC) by SO and the development of a strategy to enhance this effect. Although SO administration diminished clonogenicity,spheroid formation,aldehyde dehydrogenase 1 (ALDH1) activity,growth on immunodeficient mice,proliferation,and angiogenesis and induced apoptosis,we observed SO-induced activation of NF-kappaB associated with survival and regrowth of spheroids. For enhanced elimination of CSC characteristics by SO,we cotreated cells with sulforaphane (SF). This broccoli isothiocyanate was recently described to eliminate pancreatic CSCs by downregulation of NF-kappaB activity without inducing toxic side effects. On combination treatment,SF completely eradicated SO-induced NF-kappaB binding,which was associated with abrogated clonogenicity,spheroid formation,ALDH1 activity,migratory capacity,and induction of apoptosis. In vivo,combination therapy reduced the tumor size in a synergistic manner. This was due to induction of apoptosis,inhibition of proliferation and angiogenesis,and downregulation of SO-induced expression of proteins involved in epithelial-mesenchymal transition. Our data suggest that SF may be suited to increase targeting of CSCs by SO. View Publication

Loeys-Dietz syndrome (LDS) is an autosomal-dominant connective tissue disorder,commonly caused by genetic mutation of transforming growth factor-beta receptor (TGFBR)-1 or TGFBR2. This study describes the generation of human induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells obtained from an LDS patient with TGFBR2 mutation (R193W). Analysis confirmed the cells had a normal karyotype,expressed typical pluripotency markers,had the ability to differentiate into all three germ layers in vivo,and retained the TGFBR2 mutation from the derived hiPSCs. This iPSC line represents a potentially useful tool for investigating LDS disease mechanisms. View Publication