技术资料

Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK),a key regulator for controlling energy metabolism,is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest,without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers,Nanog and stage-specific embryonic antigen-1,in the presence of leukemia inhibitory factor,without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently,an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation,an effect inhibited by MG132,a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs,it increased expression levels of erythroid cell lineage markers (Ter119,GATA1,Klf1,Hbb-b,and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR,endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs. View Publication

Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important,given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here,we present an in-depth quantitative mass spectrometry-based analysis of hESCs,two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless,a small group of 58 proteins,mainly related to metabolism,antigen processing and cell adhesion,was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap,highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. View Publication

Efficient approaches for the precise genetic engineering of human pluripotent stem cells (hPSCs) can enhance both basic and applied stem cell research. Adeno- associated virus (AAV) vectors are of particular interest for their capacity to mediate efficient gene delivery to and gene targeting in various cells. However,natural AAV serotypes offer only modest transduction of human embryonic and induced pluripotent stem cells (hESCs and hiPSCs),which limits their utility for efficiently manipulating the hPSC genome. Directed evolution is a powerful means to generate viral vectors with novel capabilities,and we have applied this approach to create a novel AAV variant with high gene delivery efficiencies (˜50%) to hPSCs,which are importantly accompanied by a considerable increase in gene-targeting frequencies,up to 0.12%. While this level is likely sufficient for numerous applications,we also show that the gene-targeting efficiency mediated by an evolved AAV variant can be further enhanced (textgreater1%) in the presence of targeted double- stranded breaks (DSBs) generated by the co-delivery of artificial zinc finger nucleases (ZFNs). Thus,this study demonstrates that under appropriate selective pressures,AAV vectors can be created to mediate efficient gene targeting in hPSCs,alone or in the presence of ZFN- mediated double-stranded DNA breaks. View Publication

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic $\$-cells. In other systems,Musashi genes regulate cell fate via Notch signaling,which we recently showed regulates $\$-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms,as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of $\$-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1,while it decreased Ins1 and Ins2 expression,revealing a molecular link between ER stress and $\$-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1,stimulate proliferation,inhibit apoptosis and reduce insulin expression,whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together,these results demonstrate overlapping,but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and $\$-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory $\$-cell proliferation and the loss of $\$-cell gene expression seen in specific phases of the progression to type 2 diabetes. View Publication

Considerable efforts have been made since the 1950s to better understand the cellular and molecular mechanisms of action of metformin,a potent antihyperglycaemic agent now recommended as the first-line oral therapy for T2D (Type 2 diabetes). The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production,mostly through a mild and transient inhibition of the mitochondrial respiratory chain complex I. In addition,the resulting decrease in hepatic energy status activates AMPK (AMP-activated protein kinase),a cellular metabolic sensor,providing a generally accepted mechanism for the action of metformin on hepatic gluconeogenesis. The demonstration that respiratory chain complex I,but not AMPK,is the primary target of metformin was recently strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice. Beyond its effect on glucose metabolism,metformin has been reported to restore ovarian function in PCOS (polycystic ovary syndrome),reduce fatty liver,and to lower microvascular and macrovascular complications associated with T2D. Its use has also recently been suggested as an adjuvant treatment for cancer or gestational diabetes and for the prevention in pre-diabetic populations. These emerging new therapeutic areas for metformin will be reviewed together with recent findings from pharmacogenetic studies linking genetic variations to drug response,a promising new step towards personalized medicine in the treatment of T2D. View Publication

Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However,the lack of simple,robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells,2) significantly increases viability and replating efficiency,3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation,we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%,with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions,and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs. View Publication

INTRODUCTION: The expression of proinflammatory protein tissue transglutaminase 2 (TG2) is frequently upregulated in multiple cancer cell types. However,the exact role of TG2 in cancer cells is not well-understood. We recently initiated studies to determine the significance of TG2 in cancer cells and observed that sustained expression of TG2 resulted in epithelial-to-mesenchymal transition (EMT) and promoted cancer stem cell (CSC) traits in mammary epithelial cells. These results suggested that TG2 could serve as a promising therapeutic target for overcoming chemoresistance and inhibiting metastatic spread of cancer cells. METHODS: Using various mutant constructs,we analyzed the activity of TG2 that is essential for promoting the EMT-CSC phenotype. RESULTS: Our results suggest that catalytically inactive TG2 (TG2-C277S) is as effective as wild-type TG2 (TG2-WT) in inducing the EMT-CSC in mammary epithelial cells. In contrast,overexpression of a GTP-binding-deficient mutant (TG2-R580A) was completely incompetent in this regard. Moreover,TG2-dependent activation of the proinflammatory transcription factor NF-κB is deemed essential for promoting the EMT-CSC phenotype in mammary epithelial cells. CONCLUSIONS: Our results suggest that the transamidation activity of TG2 is not essential for promoting its oncogenic functions and provide a strong rationale for developing small-molecule inhibitors to block GTP-binding pockets of TG2. Such inhibitors may have great potential for inhibiting the TG2-regulated pathways,reversing drug resistance and inhibiting the metastasis of cancer cells. View Publication

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression,could be maintained in culture for several weeks,expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice,the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease View Publication

The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder,ATR-Seckel syndrome. To understand how ATR functions during neurogenesis,we conditionally deleted Atr broadly throughout the murine nervous system,or in a restricted manner in the dorsal telencephalon. Unexpectedly,in both scenarios,Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16(Ink4a/Arf))-independent proliferation arrest,other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs,in the nervous system,p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion,supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress,our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis. View Publication

Deficiency of the nuclear factor-kappa-B essential modulator (NEMO) is a rare X-linked disorder that presents in boys as hypohydrotic ectodermal dysplasia with immunodeficiency due to defective nuclear factor-κB activation. Here we report on the generation of 2 human embryonic stem cell lines from discarded in vitro fertilization (IVF) embryos ascertained via preimplantation genetic diagnosis. We have derived two human embryonic stem cell lines that carry a T458G hypomorphic mutation in exon 4 of the NEMO (or IKBKG) gene. One of the lines is diploid male; the other is diploid female but has clonally inactivated the X-chromosome that harbors the wild-type IKBKG gene. We show that both lines are pluripotent,have the capacity to differentiate into hematopoietic progenitors,and have defective inhibitor of nuclear factor kappa-B kinase activity. These NEMO deficiency hES cell lines provide an unlimited source for differentiated cell types and may serve as a unique tool to study NEMO deficiency and potentially lead to the development of new therapies for this disease. View Publication

Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced,but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5),which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9),is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However,LSD1 is not essential for the maintenance of ESC identity. Instead,ESCs lacking LSD1 activity fail to differentiate fully,and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers,LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex,which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation,which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states. View Publication

BACKGROUND Cryopreservation protocols have remained relatively unchanged since the first umbilical cord blood banking program was established. This study evaluated the preservation efficacy of a novel intracellular-like cryopreservation solution (CryoStor,BioLife Solutions,Inc.),the rate of addition of two cryopreservation solutions to cord blood units (CBUs),and reduced final dimethyl sulfoxide (DMSO) concentration of 5%. STUDY DESIGN AND METHODS Split-sample CBUs were cryopreserved with either an in-house 20% DMSO-based cryopreservation solution or CryoStor CS10 at a rate of 1 mL/min (n = 10; i.e.,slow addition) or as a bolus injection (n = 6; i.e.,fast addition). Infrared images of exothermic effects of the cryopreservation solutions were monitored relative to the rate of addition. Prefreeze and postthaw colony-forming unit assays,total nucleated cells,and CD34+ cell counts were compared. RESULTS Maximum temperature excursions observed were less than 6°C,regardless of the rate of solution addition. Fast addition resulted in peak excursions approximately twice that of slow addition but the magnitude and duration were minimal and transient. Slow addition of CryoStor CS10 (i.e.,final concentration % 5% DMSO) resulted in significantly better postthaw CD34+ cell recoveries; no other metrics were significantly different. Fast addition of CryoStor resulted in similar postthaw metrics compared to slow addition of the in-house solution. CONCLUSION Slow and fast addition of cryopreservation solutions result in mean temperature changes of approximately 3.3 to 4.45°C. Postthaw recoveries with CryoStor were equivalent to or slightly better than with the in-house cryopreservation solution. CryoStor also provides several advantages including reduced processing time,formulation consistency,and reduced DMSO in the frozen product (% 5%). View Publication