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Aberrant histone deacetylase (HDAC) activity is frequent in human leukemias. However,while classical,NAD(+)-independent HDACs are an established therapeutic target,the relevance of NAD(+)-dependent HDACs (sirtuins) in leukemia treatment remains unclear. Here,we assessed the antileukemic activity of sirtuin inhibitors and of the NAD(+)-lowering drug FK866,alone and in combination with traditional HDAC inhibitors. Primary leukemia cells,leukemia cell lines,healthy leukocytes and hematopoietic progenitors were treated with sirtuin inhibitors (sirtinol,cambinol,EX527) and with FK866,with or without addition of the HDAC inhibitors valproic acid,sodium butyrate,and vorinostat. Cell death was quantified by propidium iodide cell staining and subsequent flow-cytometry. Apoptosis induction was monitored by cell staining with FITC-Annexin-V/propidium iodide or with TMRE followed by flow-cytometric analysis,and by measuring caspase3/7 activity. Intracellular Bax was detected by flow-cytometry and western blotting. Cellular NAD(+) levels were measured by enzymatic cycling assays. Bax was overexpressed by retroviral transduction. Bax and SIRT1 were silenced by RNA-interference. Sirtuin inhibitors and FK866 synergistically enhanced HDAC inhibitor activity in leukemia cells,but not in healthy leukocytes and hematopoietic progenitors. In leukemia cells,HDAC inhibitors were found to induce upregulation of Bax,a pro-apoptotic Bcl2 family-member whose translocation to mitochondria is normally prevented by SIRT1. As a result,leukemia cells become sensitized to sirtuin inhibitor-induced apoptosis. In conclusion,NAD(+)-independent HDACs and sirtuins cooperate in leukemia cells to avoid apoptosis. Combining sirtuin with HDAC inhibitors results in synergistic antileukemic activity that could be therapeutically exploited. View Publication

We developed a protocol of in vitro differentiation of human embryonic stem cells into three-dimensional structures histologically and molecularly similar to the developing retina. View Publication

Nuclear-architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as ageing. It is therefore plausible that diseases whose manifestations correlate with ageing might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of ageing-associated disorders by focusing on a leucine-rich repeat kinase 2 (LRRK2) dominant mutation (G2019S; glycine-to-serine substitution at amino acid 2019),which is associated with familial and sporadic Parkinson's disease as well as impairment of adult neurogenesis in mice. Here we report on the generation of induced pluripotent stem cells (iPSCs) derived from Parkinson's disease patients and the implications of LRRK2(G2019S) mutation in human neural-stem-cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in nuclear-envelope organization,clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in Parkinson's disease iPSCs and were recapitulated after targeted knock-in of the LRRK2(G2019S) mutation in human embryonic stem cells. Analysis of human brain tissue showed nuclear-envelope impairment in clinically diagnosed Parkinson's disease patients. Together,our results identify the nucleus as a previously unknown cellular organelle in Parkinson's disease pathology and may help to open new avenues for Parkinson's disease diagnoses as well as for the potential development of therapeutics targeting this fundamental cell structure. View Publication

The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find,unexpectedly,that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2,3,5,6,7 and 11),whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP,whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results,hESCs contain a previously undescribed complex comprising TAFs 2,6,7,11 and TBP. Altering the composition of hESC TAFs,either by depleting TAFs that are present or ectopically expressing TAFs that are absent,results in misregulated expression of pluripotency genes and induction of differentiation. Thus,the selective expression and use of TAFs underlies the ability of hESCs to self-renew.DOI:http://dx.doi.org/10.7554/eLife.00068.001. View Publication

Skin-derived precursor (SKP) cells are a valuable resource for tissue engineering and regenerative medicine,because they represent multipotent stem cells that differentiate into neural and mesodermal progenies. Previous studies suggest that the stem cell pool decreases with age. Here,we show that human multipotent SKP cells can be efficiently collected from adult cheek/chin skin,even in aged individuals of 70-78years. SKP cells were isolated from 38 skin samples by serum-free sphere culture and examined for the ability to differentiate into neural and mesodermal lineages. The number of spheres obtained from adult facial skin was significantly higher than that of trunk or extremity skin. SKP cells derived from cheek/chin skin exhibited a high ability to differentiate into neural and mesodermal cells relative to those derived from eyelid,trunk,or extremity skin. Furthermore,cheek/chin skin SKP cells were shown to express markers for undifferentiated stem cells,including a high expression level of the Sox9 gene. These results indicate that cheek/chin skin is useful for the recovery of multipotent stem cells for tissue engineering and regenerative therapy. View Publication

Demonstrates efficient reprogramming of iPS cells from CD34+ stem cells enriched from a small volume of peripheral blood. View Publication

Label-retaining cancer cells (LRCCs) represent a novel population of stem-like cancer cells exhibiting slow cycling,chemoresistance and tumor-initiating capacities; however,their properties remain unclear,and approaches to eradicate LRCCs remain elusive. Here,we report that colon cancer cells with high fluorescent intensity,referred to as LRCCs,have the greatest cancer stem cell (CSC)-like capacities and that they preferentially express CSC markers and stemness-related genes. Moreover,we found that Lgr5,which has been reported to be a marker of rapid cycling CSCs,is almost negatively expressed in LRCCs but that its expression is gradually increased in the differentiation process of LRCCs. Interestingly,we found that LRCCs are especially sensitive to the pro-apoptotic effect of IFN-γ treatment both in vitro and in vivo because LRCCs possess higher IFN-γR levels compared with non-LRCCs,which results in the upregulation of the apoptosis pathway after IFN-γ treatment. Furthermore,we found that IFN-γ shows synergistic effects with the conventional anticancer drug Oxaliplatin to eliminate both LRCCs and non-LRCCs. In conclusion,this is the first study to suggest that LRCCs,as a distinct tumor-initiating population,can be selectively eradicated by IFN-γ,which may provide a novel therapeutic strategy for colon cancer treatment. View Publication

Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling,which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs,suggesting that they may influence self-renewal. To test this hypothesis,we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation,whereas SPRY4 KD enhanced survival. In both cases,after KD the cells were able to differentiate into cells of the three germ layers,although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage,explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs. View Publication

Background: Human induced pluripotent stem cells,which can be differentiated into hepatocyte-like cells,could provide a source for liver regeneration and bio-artificial liver devices. However,the functionality of hepatocyte-like cells is significantly lower than that of primary hepatocytes. Aims: To investigate whether serum from patients undergoing hepatectomy might promote differentiation from human induced pluripotent stem cells to hepatocyte-like cells. Methods: Serum from patients undergoing hepatectomy (acquired pre-hepatectomy and 3. hours,1 day and 3 days post-hepatectomy) was used to replace foetal bovine serum when differentiating human induced pluripotent stem cells into hepatocyte-like cells. Properties of hepatocyte-like cells were assessed and compared with cells cultured in foetal bovine serum. Results: The differentiation efficiency and functionality of hepatocyte-like cells cultured in human serum 3. hours and 1 day post-hepatectomy were superior to those cultured in foetal bovine serum and human serum pre-hepatectomy. Human serum 3 days post-hepatectomy had an equal effect to that of human serum pre-hepatectomy. Some cytochrome P450 isozyme transcript levels of hepatocyte-like cells cultured in human serum were higher than those cultured in foetal bovine serum. Conclusion: Human serum,particularly that acquired relatively soon after hepatectomy,can enhance the differentiation efficiency and functionality of hepatocyte-like cells derived from human induced pluripotent stem cells. textcopyright 2014 Editrice Gastroenterologica Italiana S.r.l. View Publication

Molecular beacons (MBs) are dual-labeled oligonucleotides that fluoresce only in the presence of complementary mRNA. The use of MBs to target specific mRNAs allows sorting of specific cells from a mixed cell population. In contrast to existing approaches that are limited by available surface markers or selectable metabolic characteristics,the MB-based method enables the isolation of a wide variety of cells. For example,the ability to purify specific cell types derived from pluripotent stem cells (PSCs) is important for basic research and therapeutics. In addition to providing a general protocol for MB design,validation and nucleofection into cells,we describe how to isolate a specific cell population from differentiating PSCs. By using this protocol,we have successfully isolated cardiomyocytes differentiated from mouse or human PSCs (hPSCs) with ∼ 97% purity,as confirmed by electrophysiology and immunocytochemistry. After designing MBs,their ordering and validation requires 2 weeks,and the isolation process requires 3 h. View Publication