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We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27,a key regulator of cell cycle,in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly,expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly,when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients,increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition,when CLL cells were induced in vitro to enter cell cycle (eg,with cytosine phosphate guanine oligodeoxynucleotide),a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition. View Publication

Rearrangement of the actin cytoskeleton is essential for dynamic cellular processes. Decreased actin turnover and rigidity of cytoskeletal structures have been associated with aging and cell death. Gelsolin is a Ca(2+)-activated actin-severing protein that is widely expressed throughout the adult mammalian brain. Here,we used gelsolin-deficient (Gsn(-/-)) mice as a model system for actin filament stabilization. In Gsn(-/-) mice,emigration of newly generated cells from the subventricular zone into the olfactory bulb was slowed. In vitro,gelsolin deficiency did not affect proliferation or neuronal differentiation of adult neural progenitors cells (NPCs) but resulted in retarded migration. Surprisingly,hippocampal neurogenesis was robustly induced by gelsolin deficiency. The ability of NPCs to intrinsically sense excitatory activity and thereby implement coupling between network activity and neurogenesis has recently been established. Depolarization-induced [Ca(2+)](i) increases and exocytotic neurotransmitter release were enhanced in Gsn(-/-) synaptosomes. Importantly,treatment of Gsn(-/-) synaptosomes with mycotoxin cytochalasin D,which,like gelsolin,produces actin disassembly,decreased enhanced Ca(2+) influx and subsequent exocytotic norepinephrine release to wild-type levels. Similarly,depolarization-induced glutamate release from Gsn(-/-) brain slices was increased. Furthermore,increased hippocampal neurogenesis in Gsn(-/-) mice was associated with a special microenvironment characterized by enhanced density of perfused vessels,increased regional cerebral blood flow,and increased endothelial nitric oxide synthase (NOS-III) expression in hippocampus. Together,reduced filamentous actin turnover in presynaptic terminals causes increased Ca(2+) influx and,subsequently,elevated exocytotic neurotransmitter release acting on neural progenitors. Increased neurogenesis in Gsn(-/-) hippocampus is associated with a special vascular niche for neurogenesis. View Publication

We report here that butyrate,a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent,greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment,the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (textgreater100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines,including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors,show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation,we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming,butyrate treatment enhanced histone H3 acetylation,promoter DNA demethylation,and the expression of endogenous pluripotency-associated genes,including DPPA2,whose overexpression partially substitutes for butyrate stimulation. Thus,butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover,butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells,including cells from patients that are more refractory to reprogramming. View Publication

We have derived 30 human embryonic stem cell lines from supernumerary blastocysts in our laboratory. During the derivation process,we have studied new and safe method to establish good quality lines. All our human embryonic stem cell lines have been derived using human foreskin fibroblasts as feeder cells. The 26 more recent lines were derived in a medium containing serum replacement instead of fetal calf serum. Mechanical isolation of the inner cell mass using flexible metal needles was used in deriving the 10 latest lines. The lines are karyotypically normal,but culture adaptation in two lines has been observed. Our human embryonic stem cell lines are banked,and they are available for researchers. View Publication

Mesenchymal stem cells (MSCs) have multilineage differentiation potential which includes cell lineages of the central nervous system; hence MSCs might be useful in the treatment of neurodegenerative diseases such as Parkinson's disease. Although mesenchymal stem cells have been shown to differentiate into the neural lineage,there is still little knowledge about the underlying mechanisms of differentiation particularly towards specialized neurons such as dopaminergic neurons. Here,we show that MSCs derived from human umbilical cord blood (MSC(hUCBs)) are capable of expressing tyrosine hydroxylase (TH) and Nurr1,markers typically associated with DA neurons. We also found differential phosphorylation of TH isoforms indicating the presence of post-translational mechanisms possibly activating and modifying TH in MSC(hUCB). Furthermore,functional dissection of components in the differentiation medium revealed that dibutyryl-cAMP (db-cAMP),3-isobutyl-1-methylxanthine (IBMX) and retinoic acid (RA) are involved in the regulation of Nurr1 and Neurofilament-L expression as well as in the differential phosphorylation of TH. We also demonstrate a possible inhibitory role of the protein kinase A signaling pathway in the phosphorylation of specific TH isoforms. View Publication

Breast cancer stem cells are a group of undifferentiated cells with self-renewal and multidifferentiation potential. Chemotherapeutic and radiotherapeutic resistance,hypoxic resistance,high tumorigenicity,high cell invasion,and metastatic abilities are characteristics of these cells,which are responsible for breast cancer recurrence. Therefore,the correct sorting and identification of breast cancer stem cells is a primary step for research in this field. This article briefly describes the recent progress on sorting and identification technologies for breast cancer stem cells. Sorting technologies include the side population technique,technologies that depend on cell surface markers,ALDEFLUOR assays,and in situ detection. Identification technologies include mammosphere cultures,limited dilution in vitro,and in-vivo animal models. This review provides an important reference for breast cancer stem cell research,which will explore new methods for the treatment of patients with breast cancer. View Publication

GCS-100 is a galectin-3 antagonist with an acceptable human safety profile that has been demonstrated to have an antimyeloma effect in the context of bortezomib resistance. In the present study,the mechanisms of action of GCS-100 are elucidated in myeloma cell lines and primary tumor cells. GCS-100 induced inhibition of proliferation,accumulation of cells in sub-G(1) and G(1) phases,and apoptosis with activation of both caspase-8 and -9 pathways. Dose- and time-dependent decreases in MCL-1 and BCL-X(L) levels also occurred,accompanied by a rapid induction of NOXA protein,whereas BCL-2,BAX,BAK,BIM,BAD,BID,and PUMA remained unchanged. The cell-cycle inhibitor p21(Cip1) was up-regulated by GCS-100,whereas the procycling proteins CYCLIN E2,CYCLIN D2,and CDK6 were all reduced. Reduction in signal transduction was associated with lower levels of activated IkappaBalpha,IkappaB kinase,and AKT as well as lack of IkappaBalpha and AKT activation after appropriate cytokine stimulation (insulin-like growth factor-1,tumor necrosis factor-alpha). Primary myeloma cells showed a direct reduction in proliferation and viability. These data demonstrate that the novel therapeutic molecule,GCS-100,is a potent modifier of myeloma cell biology targeting apoptosis,cell cycle,and intracellular signaling and has potential for myeloma therapy. View Publication

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support,but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study,five separate laboratories,each with experience in human embryonic stem cell culture,used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods,with propagation in the presence of Knockout Serum Replacer,FGF-2,and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment,death,and differentiated morphology by phase contrast microscopy,for growth by serial cell counts,and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems,only the control and those based on two commercial media,mTeSR1 and STEMPRO,supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment,cell death,or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study,and the lack of success with other formulations from academic groups compared to previously published results,include: the complex combination of growth factors present in the commercial preparations; improved development,manufacture,and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. View Publication

PURPOSE: In vitro sensitivity to the proapoptotic receptor agonists dulanermin (rhApo2L/TRAIL) and drozitumab (DR5-agonist antibody) is strongly predicted by the expression of the O-glycosylation enzymes GALNT14 in non-small cell lung cancer (NSCLC) cell lines (among others) and of FUT3/6 in colorectal cancer (CRC) cell lines. We developed immunohistochemistry (IHC) assays that measure GALNT14 and FUT3/6 levels in archival formalin-fixed,paraffin-embedded human tumor tissue to determine marker prevalence in NSCLC and CRC tissue and to enable the future examination of these markers in clinical trials. EXPERIMENTAL DESIGN: GALNT14 or FUT3/6 ELISA-positive hybridoma clones were screened through IHC on cell pellets with known mRNA levels. The specificity of staining was examined in cell lines,normal tissue,and tumor tissue. RESULTS: GALNT14 and FUT3/6 IHC exhibited a golgi staining pattern and correlated with GALNT14 and FUT3/6 (but not GALNT2 and FUT4) mRNA expression levels in cell lines and normal tissues,suggesting specificity. GALNT14 and FUT3/6 H-scores were significantly higher in cell lines sensitive to dulanermin (P = 0.01 and P = 0.0004,respectively) and drozitumab (P = 0.03 and P textless 0.0001,respectively) versus resistant cell lines. GALNT14 and FUT3/6 H-scores varied widely,with approximately 45% of NSCLC samples exhibiting weak to moderate GALNT14 staining (H-score of at least 25) and 70% of CRC samples exhibiting moderate to strong FUT3/6 staining (H-score of at least 125). CONCLUSIONS: GALNT14 and FUT3/6 expression can be assessed in human tumors using sensitive and specific IHC assays. Both assays are being deployed in ongoing clinical trials of dulanermin and drozitumab to assess potential utility for patient selection. View Publication

We had earlier reported the derivation and characterization of two new sibling human embryonic stem cell lines BJNhem19 and BJNhem20,from discarded grade III embryos of Indian origin. We report here the characteristics of the two sibling cell lines after long-term continuous culture for over 2 yr during which they have been passaged over 200 times. We show that both cell lines adapt well to culture on various mouse and human feeders as well as in feeder-free conditions. The cells show normal diploid karyotype and continue to express all pluripotency markers. Both cell lines differentiate to derivatives of all three germ layers in vitro. However as reported earlier,BJNhem19 is unable to generate teratomas in nude or SCID mice or differentiate to beating cardiomyocytes when tested over several passages during long-term stable culture. On the other hand,the cardiac differentiation capacity of BJNhem20 is greatly increased,and it can generate beating cardiomyocytes that proliferate when isolated and cultured further. In conclusion,the two cell lines have maintained a stable phenotype for over 2 yr and are indeed immortal. Their derivation from grade III embryos does not seem to have any adverse effect on their long-term phenotype. The cells can be obtained for research purposes from the UK Stem Cell Bank and from the authors. View Publication

A large number of human embryonic stem cell (hESC) lines have been derived worldwide since the first hESC line establishment in 1998. Despite many common characteristics,most important of which is the pluripotency,hESC lines vary significantly in their transcriptional profiles,genetic,and epigenetic state. These differences may arise both from individual genetics of the cell lines and from variations in their handling such as isolation and cultivation. In order to minimize the latter differences,the standardized protocols of cultivation and inter-laboratory comprehensive studies should be performed. In this report,we summarized our experience of derivation and characterization of hESC lines as well as of adaptation of hESCs to novel cultivation protocols. We have successfully derived five hESC lines and characterized them by previously established criteria,including expression of specific markers and the capacity to differentiate both in vitro and in vivo. Four of these lines,namely hESM01-04,were initially derived using mouse fibroblasts as a feeder and currently are maintained under feeder-free,serum-free conditions using mTeSR1 and Matrigel. The fifth line,hESMK05 was derived in feeder-free,serum-free conditions using mTeSR1 and Matrigel. Cell lines retain their pluripotent status and normal karyotype for more than 70 passages and are available to the scientific community. View Publication

A series of chalcones (1-9) and pyrazoles (10-18) was prepared to investigate their potential activity as Angiotensin I-Converting Enzyme (ACE) inhibitors. Their structures were verified by elemental analysis,UV,IR,MS,(1)H NMR,(13)C NMR,and 2D NMR experiments. Among tested compounds,chalcone 7 exerted the highest activity with an IC(50) value of 0.219 mM,while the most potent pyrazole was 15 (IC(50) value of 0.213 mM). View Publication