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High aldehyde dehydrogenase (ALDH) activity is a marker commonly used to isolate stem cells,particularly breast cancer stem cells (CSCs). Here,we determined that ALDH1A1 activity is inhibited by acetylation of lysine 353 (K353) and that acetyltransferase P300/CBP-associated factor (PCAF) and deacetylase sirtuin 2 (SIRT2) are responsible for regulating the acetylation state of ALDH1A1 K353. Evaluation of breast carcinoma tissues from patients revealed that cells with high ALDH1 activity have low ALDH1A1 acetylation and are capable of self-renewal. Acetylation of ALDH1A1 inhibited both the stem cell population and self-renewal properties in breast cancer. Moreover,NOTCH signaling activated ALDH1A1 through the induction of SIRT2,leading to ALDH1A1 deacetylation and enzymatic activation to promote breast CSCs. In breast cancer xenograft models,replacement of endogenous ALDH1A1 with an acetylation mimetic mutant inhibited tumorigenesis and tumor growth. Together,the results from our study reveal a function and mechanism of ALDH1A1 acetylation in regulating breast CSCs. View Publication

The bacterial CRISPR-Cas9 system has emerged as an effective tool for sequence-specific gene knockout through non-homologous end joining (NHEJ),but it remains inefficient for precise editing of genome sequences. Here we develop a reporter-based screening approach for high-throughput identification of chemical compounds that can modulate precise genome editing through homology-directed repair (HDR). Using our screening method,we have identified small molecules that can enhance CRISPR-mediated HDR efficiency,3-fold for large fragment insertions and 9-fold for point mutations. Interestingly,we have also observed that a small molecule that inhibits HDR can enhance frame shift insertion and deletion (indel) mutations mediated by NHEJ. The identified small molecules function robustly in diverse cell types with minimal toxicity. The use of small molecules provides a simple and effective strategy to enhance precise genome engineering applications and facilitates the study of DNA repair mechanisms in mammalian cells. View Publication

Hypothalamic neurons orchestrate many essential physiological and behavioral processes via secreted neuropeptides,and are relevant to human diseases such as obesity,narcolepsy and infertility. We report the differentiation of human pluripotent stem cells into many of the major types of neuropeptidergic hypothalamic neurons,including those producing pro-opiolemelanocortin,agouti-related peptide,hypocretin/orexin,melanin-concentrating hormone,oxytocin,arginine vasopressin,corticotropin-releasing hormone (CRH) or thyrotropin-releasing hormone. Hypothalamic neurons can be generated using a 'self-patterning' strategy that yields a broad array of cell types,or via a more reproducible directed differentiation approach. Stem cell-derived human hypothalamic neurons share characteristic morphological properties and gene expression patterns with their counterparts in vivo,and are able to integrate into the mouse brain. These neurons could form the basis of cellular models,chemical screens or cellular therapies to study and treat common human diseases. View Publication

OBJECTIVE: Differentiation of human embryonic stem (hES) cells to fully developed cell types holds great therapeutic promise. Despite significant progress,the conversion of hES cells to stable,fully differentiated endocrine cells that exhibit physiologically regulated hormone secretion has not yet been achieved. Here we describe an efficient differentiation protocol for the in vitro conversion of hES cells to functional glucagon-producing α- cells. RESEARCH DESIGN AND METHODS: Using a combination of small molecule screening and empirical testing,we developed a six-stage differentiation protocol for creating functional α-cells. An extensive in vitro and in vivo characterization of the differentiated cells was performed. RESULTS: A high rate of synaptophysin expression (textgreater75%) and robust expression of glucagon and the α-cell transcription factor ARX was achieved. After a transient polyhormonal state in which cells coexpress glucagon and insulin,maturation in vitro or in vivo resulted in depletion of insulin and other β-cell markers with concomitant enrichment of α-cell markers. After transplantation,these cells secreted fully processed,biologically active glucagon in response to physiologic stimuli including prolonged fasting and amino acid challenge. Moreover,glucagon release from transplanted cells was sufficient to reduce demand for pancreatic glucagon,resulting in a significant decrease in pancreatic α-cell mass. CONCLUSIONS: These results indicate that fully differentiated pancreatic endocrine cells can be created via stepwise differentiation of hES cells. These cells may serve as a useful screening tool for the identification of compounds that modulate glucagon secretion as well as those that promote the transdifferentiation of α-cells to β-cells. View Publication

Clinical implications of induced pluripotent stem (iPS) cell technology are enormous for personalized medicine. However,extensive use of viral approach for ectopic expression of reprogramming factors is a major hurdle in realization of its true potential. Non-viral methods for making iPS cells,although plausible,are impractical because of high cost. MicroRNAs are important cellular modulators that have been shown to rival transcription factors and are important players in embryonic development. We have generated distinct microRNA-omes" signature of iPS cells that remain in a near embryonic stem (ES) cell state and distinct from differentiated cells. Recent advances in the microRNA field and experimentally validated microRNAs warrant a review in experimental protocols for microRNA expression profile." View Publication

Current evidence suggests that much like leukemia,breast tumors are maintained by a small subpopulation of tumor cells that have stem cell properties. These cancer stem cells are envisaged to be responsible for tumor formation and relapse. Therefore,knowledge about their nature will provide a platform to develop therapies to eliminate these breast cancer stem cells. This concept highlights the need to understand the mechanisms that regulate the normal functions of the breast stem cells and their immediate progeny as alterations to these same mechanisms can cause these primitive cells to act as cancer stem cells. The study of the primitive cell functions relies on the ability to isolate them from primary sources of breast tissue. This chapter describes processing of discarded tissue from reduction mammoplasty samples as sources of normal primary human breast epithelial cells and describes cell culture systems to grow single-cell suspensions prepared from these reduction samples in vitro. View Publication

We report for the first time a microdevice that enables the selective enrichment,culture,and identification of tumor-initiating cells on native polydimethylsiloxane (PDMS). For nearly a decade,researchers have identified tumor-initiating breast cancer cells within heterogeneous populations of breast cancer cells by utilizing low-attachment serum-free culture conditions,which lead to the formation of spheroidal colonies (mammospheres) that are enriched for tumor-initiating cells. However,the utility of this assay has been limited by difficulties in combining this culture-plate-based technique with other cellular and molecular analyses. Integrating the mammosphere technique into a microsystem can enable it to be combined directly with a number of functions,such as cell sorting,drug screens,and molecular assays. In this work,we demonstrate mammosphere culture within a PDMS microdevice. We first prove that a native hydrophobic PDMS surface is as effective as commercial low-attachment plates at selectively promoting the formation of mammospheres. We then experimentally assess the PDMS microdevice. Time-lapse images of mammosphere formation within the microdevice show that mammospheres form from single cells or small clusters of cells. Following formation of the mammospheres,it is desirable to evaluate the cells within the spheroids for enrichment of tumor initiating cells. To perform assays such as this (which require the loading and rinsing of reagents) without flushing the cells (which are in suspension) from the device,the culture chamber is separated from a reagent reservoir by a commercially available microporous membrane,and thus reagents are exchanged between the reservoir and the culture chamber by diffusion only. Using this capability,we verify that the mammospheres are enriched for tumor initiating cells by staining aldehyde dehydrogenase activity,a cancer stem cell marker. To the best of our knowledge,this is the first assay that enables the direct observation of tumor-initiating cells within a suspended mammosphere. View Publication

ATP citrate lyase (ACL) knockdown (KD) causes tumor suppression and induces differentiation. We have previously reported that ACL KD reverses epithelial-mesenchymal transition (EMT) in lung cancer cells. Because EMT is often associated with processes that induce stemness,we hypothesized that ACL KD impacts cancer stem cells. By assessing tumorsphere formation and expression of stem cell markers,we showed this to be the case in A549 cells,which harbor a Ras mutation,and in two other non-small-cell lung cancer cell lines,H1975 and H1650,driven by activating EGFR mutations. Inducible ACL KD had the same effect as stable ACL KD. Similar effects were noted in another well-characterized Ras-induced mammary model system (HMLER). Moreover,treatment with hydroxycitrate phenocopied the effects of ACL KD,suggesting that the enzymatic activity of ACL was critical. Indeed,acetate treatment reversed the ACL KD phenotype. Having previously established that ACL KD impacts signaling through the phosphatidylinositol 3-kinase (PI3K) pathway,not the Ras-mitogen-activated protein kinase (MAPK) pathway,and that EMT can be reversed by PI3K inhibitors,we were surprised to find that stemness in these systems was maintained through Ras-MAPK signaling,and not via PI3K signaling. Snail is a downstream transcription factor impacted by Ras-MAPK signaling and known to promote EMT and stemness. We found that snail expression was reduced by ACL KD. In tumorigenic HMLER cells,ACL overexpression increased snail expression and stemness,both of which were reduced by ACL KD. Furthermore,ACL could not initiate either tumorigenesis or stemness by itself. ACL and snail proteins interacted and ACL expression regulated the transcriptional activity of snail. Finally,ACL KD counteracted stem cell characteristics induced in diverse cell systems driven by activation of pathways outside of Ras-MAPK signaling. Our findings unveil a novel aspect of ACL function,namely its impact on cancer stemness in a broad range of genetically diverse cell types. View Publication

The advent of human induced pluripotent stem cell (hiPSC) technology has produced patient-specific hiPSC derived cardiomyocytes (hiPSC-CMs) that can be used as a platform to study cardiac diseases and to explore new therapies.The ability to genetically manipulate hiPSC-CMs not only is essential for identifying the structural and/or functional role of a protein but can also provide valuable information regarding therapeutic applications. In this chapter,we describe protocols for culture,maintenance,and cardiac differentiation of hiPSCs. Then,we provide a basic procedure to transduce hiPSC-CMs. View Publication

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells,which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered,the rank order of potency is oleoyl-LPA textgreater arachidonoyl-LPA textgreater linolenoyl-LPA textgreater linoleoyl-LPA textgreater stearoyl-LPA = palmitoyl-LPA textgreater myristoyl-LPA. The shorter-chain species,lauroyl- and decanoyl-LPA,show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses textgreater 15 microM. Similarly,the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate,a putative messenger with some structural similarities to LPA,elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however,cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that,although many features of the LPA structure are important for optimal activity,the phosphate group is most critical,suggesting that this moiety is directly involved in receptor activation. View Publication

MCF-7/AdrVp is a multidrug-resistant human breast cancer subline that displays an ATP-dependent reduction in the intracellular accumulation of anthracycline anticancer drugs in the absence of overexpression of known multidrug resistance transporters such as P glycoprotein or the multidrug resistance protein. RNA fingerprinting led to the identification of a 2.4-kb mRNA that is overexpressed in MCF-7/AdrVp cells relative to parental MCF-7 cells. The mRNA encodes a 655-aa [corrected] member of the ATP-binding cassette superfamily of transporters that we term breast cancer resistance protein (BCRP). Enforced expression of the full-length BCRP cDNA in MCF-7 breast cancer cells confers resistance to mitoxantrone,doxorubicin,and daunorubicin,reduces daunorubicin accumulation and retention,and causes an ATP-dependent enhancement of the efflux of rhodamine 123 in the cloned transfected cells. BCRP is a xenobiotic transporter that appears to play a major role in the multidrug resistance phenotype of MCF-7/AdrVp human breast cancer cells. View Publication

The intestine is maintained by stem cells located at the base of crypts and distinguished by the expression of LGR5. Genetically engineered mouse models have provided a wealth of information about intestinal stem cells,whereas less is known about human intestinal stem cells owing to difficulty detecting and isolating these cells. We established an organoid repository from patient-derived adenomas,adenocarcinomas and normal colon,which we analyzed for variants in 71 colorectal cancer (CRC)-associated genes. Normal and neoplastic colon tissue organoids were analyzed by immunohistochemistry and fluorescent-activated cell sorting for LGR5. LGR5-positive cells were isolated from four adenoma organoid lines and were subjected to RNA sequencing. We found that LGR5 expression in the epithelium and stroma was associated with tumor stage,and by integrating functional experiments with LGR5-sorted cell RNA sequencing data from adenoma and normal organoids,we found correlations between LGR5 and CRC-specific genes,including dickkopf WNT signaling pathway inhibitor 4 (DKK4) and SPARC-related modular calcium binding 2 (SMOC2). Collectively,this work provides resources,methods and new markers to isolate and study stem cells in human tissue homeostasis and carcinogenesis. View Publication