Shuhua Chen

Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes

BackgroundCharacterization of gene expression signatures for cardiomyocytes derived from embryonic stem cells will help to define their early biologic processes.ResultsA transgenic α-myosin heavy chain (MHC) embryonic stem cell lineage was generated, exhibiting puromycin resistance and expressing enhanced green fluorescent protein (EGFP) under the control of the α-MHC promoter. A puromycin-resistant, EGFP-positive, α-MHC-positive cardiomyocyte population was isolated with over 92% purity. RNA was isolated after electrophysiological characterization of the cardiomyocytes. Comprehensive transcriptome analysis of α-MHC-positive cardiomyocytes in comparison with undifferentiated α-MHC embryonic stem cells and the control population from 15-day-old embryoid bodies led to identification of 884 upregulated probe sets and 951 downregulated probe sets in α-MHC-positive cardiomyocytes. A subset of upregulated genes encodes cytoskeletal and voltage-dependent channel proteins, and proteins that participate in aerobic energy metabolism. Interestingly, mitosis, apoptosis, and Wnt signaling-associated genes were downregulated in the cardiomyocytes. In contrast, annotations for genes upregulated in the α-MHC-positive cardiomyocytes are enriched for the following Gene Ontology (GO) categories: enzyme-linked receptor protein signaling pathway (GO:0007167), protein kinase activity (GO:0004672), negative regulation of Wnt receptor signaling pathway (GO:0030178), and regulation of cell size (O:0008361). They were also enriched for the Biocarta p38 mitogen-activated protein kinase signaling pathway and Kyoto Encyclopedia of Genes and Genomes (KEGG) calcium signaling pathway.ConclusionThe specific pattern of gene expression in the cardiomyocytes derived from embryonic stem cells reflects the biologic, physiologic, and functional processes that take place in mature cardiomyocytes. Identification of cardiomyocyte-specific gene expression patterns and signaling pathways will contribute toward elucidating their roles in intact cardiac function.

Therapeutic targeting of a robust non-oncogene addiction to PRKDC in ATM-defective tumors.

Treating ATM-deficient cancers with an inhibitor of DNA-PKcs induces apoptosis due to inability to repair double-strand breaks in DNA. Two Wrongs Making a Right for Cancer Treatment When a cell’s DNA is damaged, its normal response is to repair its DNA or undergo apoptosis, a programmed death for cells that are damaged beyond repair. Cancer cells don’t always undergo apoptosis when they should and thus accumulate mutations over time. Even cancer cells, however, need to have some way to repair DNA damage, particularly double-strand breaks, to survive. The two normal mechanisms for such repair are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR requires the function of ATM, a kinase that’s frequently mutated in cancer cells. NHEJ is a more error-prone pathway that does not require ATM but does require another kinase, DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Now, Riabinska et al. show a way to target ATM-mutant cancer by taking advantage of the cells’ need to repair double-strand breaks in DNA. The inhibition of DNA-PKcs in cancers that were already deficient in ATM proved to be very effective for forcing them to undergo apoptosis because they could no longer repair double-strand breaks in DNA at all. DNA-PKcs inhibition did not kill normal cells or cancer cells that had a functioning HR pathway. Thus far, the effects of treating ATM-deficient tumors with DNA-PKcs inhibitors have only been shown in cultured cells and in mice, so this approach still needs to be tested in human patients. This may happen soon because one such inhibitor is already in clinical trials. In the meantime, it looks like making things go wrong in two different DNA repair pathways may yet be the right approach for treating some cancers. When the integrity of the genome is threatened, cells activate a complex, kinase-based signaling network to arrest the cell cycle, initiate DNA repair, or, if the extent of damage is beyond repair capacity, induce apoptotic cell death. The ATM protein lies at the heart of this signaling network, which is collectively referred to as the DNA damage response (DDR). ATM is involved in numerous DDR-regulated cellular responses—cell cycle arrest, DNA repair, and apoptosis. Disabling mutations in the gene encoding ATM occur frequently in various human tumors, including lung cancer and hematological malignancies. We report that ATM deficiency prevents apoptosis in human and murine cancer cells exposed to genotoxic chemotherapy. Using genetic and pharmacological approaches, we demonstrate in vitro and in vivo that ATM-defective cells display strong non-oncogene addiction to DNA-PKcs (DNA-dependent protein kinase catalytic subunit). Further, this dependence of ATM-defective cells on DNA-PKcs offers a window of opportunity for therapeutic intervention: We show that pharmacological or genetic abrogation of DNA-PKcs in ATM-defective cells leads to the accumulation of DNA double-strand breaks and the subsequent CtBP-interacting protein (CtIP)–dependent generation of large single-stranded DNA (ssDNA) repair intermediates. These ssDNA structures trigger proapoptotic signaling through the RPA/ATRIP/ATR/Chk1/p53/Puma axis, ultimately leading to the apoptotic demise of ATM-defective cells exposed to DNA-PKcs inhibitors. Finally, we demonstrate that DNA-PKcs inhibitors are effective as single agents against ATM-defective lymphomas in vivo. Together, our data implicate DNA-PKcs as a drug target for the treatment of ATM-defective malignancies.

AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis.

Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis‐antagonizing transcription factor (AATF)/Che‐1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53‐driven expression of these pro‐apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF‐depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho‐mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53‐proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53‐proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53‐driven apoptosis and commend this pathway as a target for DNA damage‐sensitizing therapeutic regimens.

Functional characterization and transcriptome analysis of embryonic stem cell-derived contractile smooth muscle cells.

Complete transcriptome profiling of contractile smooth muscle cells (SMCs) differentiated from embryonic stem cells is crucial for the characterization of smooth muscle gene expression signatures and will contribute to defining biological and physiological processes in these cells. We have generated a transgenic embryonic stem cell line expressing both the puromycin acetyl transferase and enhanced green fluorescent protein cassettes under the control of the Acta2 promoter. Applying a specific monolayer culture protocol using retinoic acid, a puromycin-resistant and enhanced green fluorescent protein–positive Acta2+ SMC population of 95% purity was isolated. Acta2+ SMCs were characterized by semiquantitative and quantitative RT-PCR profiling of SMC markers and by microarray expression profiling, as well as by immunostaining for SMC-specific cytoskeletal proteins. Patch-clamp electrophysiological characterization of these cells identified SMC-specific channels such as the ATP-sensitive potassium channel and the Ca2+-activated potassium channel. Culturing of Acta2+ SMCs in serum-containing medium resulted in a significant number of hypertrophic and binucleated cells failing to complete cell division. Functional characterization of the cells has been proved by stimulation of the cells with vasoactive agents, such as angiotensin II and endothelin. We concluded that our embryonic stem cell–derived SMC population possesses the contractile and hypertrophic phenotype of SMCs incapable of proliferation. This is the first study describing the complete transcriptome of ES-derived SMCs allowing identification of specific biological and physiological processes in the contractile phenotype SMCs and will contribute to the understanding of these processes in early SMCs derived from embryonic stem cells.

Transcriptomic and phenotypic analysis of murine embryonic stem cell derived BMP2 + lineage cells: an insight into mesodermal patterning

BackgroundBone morphogenetic protein (BMP)2 is a late mesodermal marker expressed during vertebrate development and plays a crucial role in early embryonic development. The nature of the BMP2-expressing cells during the early stages of embryonic development, their transcriptome and cell phenotypes developed from these cells have not yet been characterized.ResultsWe generated a transgenic BMP2 embryonic stem (ES) cell lineage expressing both puromycin acetyltransferase and enhanced green fluorescent protein (EGFP) driven by the BMP2 promoter. Puromycin resistant and EGFP positive BMP2+ cells with a purity of over 93% were isolated. Complete transcriptome analysis of BMP2+ cells in comparison to the undifferentiated ES cells and the control population from seven-day-old embryoid bodies (EBs; intersection of genes differentially expressed between undifferentiated ES cells and BMP2+ EBs as well as differentially expressed between seven-day-old control EBs and BMP2+ EBs by t-test, p < 0.01, fold change >2) by microarray analysis led to identification of 479 specifically upregulated and 193 downregulated transcripts. Transcription factors, apoptosis promoting factors and other signaling molecules involved in early embryonic development are mainly upregulated in BMP2+ cells. Long-term differentiation of the BMP2+ cells resulted in neural crest stem cells (NCSCs), smooth muscle cells, epithelial-like cells, neuronal-like cells, osteoblasts and monocytes. Interestingly, development of cardiomyocytes from the BMP2+ cells requires secondary EB formation.ConclusionThis is the first study to identify the complete transcriptome of BMP2+ cells and cell phenotypes from a mesodermal origin, thus offering an insight into the role of BMP2+ cells during embryonic developmental processes in vivo.

Evolutionary link between metazoan RHIM motif and prion-forming domain of fungal heterokaryon incompatibility factor HET-s/HET-s

The Rip homotypic interaction motif (RHIM) is a short, non-globular sequence stretch that mediates a key interaction of mammalian necroptosis signaling. In order to understand its unusual oligomerization properties, we set out to trace the evolutionary origins of the RHIM motif by identifying distantly related protein motifs that might employ the same binding mode. The RHIM motif was found to be related to the prion-forming domain of the HET-s protein, which oligomerizes by forming structurally well-characterized fibrils and is involved in fungal heterokaryon incompatibility. This evolutionary relationship explains the recently reported propensity of mammalian RHIM motifs to form amyloid fibrils, but suggests that these fibrils have a different structural architecture than currently assumed. These findings, together with numerous observations of RHIM-like motifs in immunity proteins from a wide range of species, provide insight to the modern innate immunity pathways in animals, plants and fungi.

Transcriptional profiling of CD31(+) cells isolated from murine embryonic stem cells.

Identification of genes involved in endothelial differentiation is of great interest for the understanding of the cellular and molecular mechanisms involved in the development of new blood vessels. Mouse embryonic stem (mES) cells serve as a potential source of endothelial cells for transcriptomic analysis. We isolated endothelial cells from 8‐days old embryoid bodies by immuno‐magnetic separation using platelet endothelial cell adhesion molecule‐1 (also known as CD31) expressed on both early and mature endothelial cells. CD31+ cells exhibit endothelial‐like behavior by being able to incorporate DiI‐labeled acetylated low‐density lipoprotein as well as form tubular structures on matrigel. Quantitative and semi‐quantitative PCR analysis further demonstrated the increased expression of endothelial transcripts. To ascertain the specific transcriptomic identity of the CD31+ cells, large‐scale microarray analysis was carried out. Comparative bioinformatic analysis reveals an enrichment of the gene ontology categories angiogenesis, blood vessel morphogenesis, vasculogenesis and blood coagulation in the CD31+ cell population. Based on the transcriptomic signatures of the CD31+ cells, we conclude that this ES cell‐derived population contains endothelial‐like cells expressing a mesodermal marker BMP2 and possess an angiogenic potential. The transcriptomic characterization of CD31+ cells enables an in vitro functional genomic model to identify genes required for angiogenesis.

Isolation and Functional Characterization of α-Smooth Muscle Actin Expressing Cardiomyocytes from Embryonic Stem Cells

Early mammalian heart development is characterized by transient expression of Α-smooth muscle actin (Acta2). To date, cardiomyocytes expressing Acta2 in the early stages of in vivo development have not been characterized. To functionally characterize Acta2-expressing cardiomyocytes, we used a transgenic ES cell line expressing both the puromycin acetyl transferase (Pac) and enhanced green fluorescent protein (EGFP) cassettes under the control of the Acta2 promoter. The onset of Acta2 expression occurred in parallel with the appearance of beating areas, indicating the formation of cardiomyocytes. Antibiotic selection resulted in a high yield of cardiomyocytes and smooth muscle cells. The green fluorescent beating areas stained positively for multiple cardiomyocyte markers. Comparative electrophysiological analysis including fetal and Α-MHC-expressing ES cell-derived cardiomyocyte controls showed that Acta2-positive cardiomyocytes contained pacemaker-, atrial- and ventricular-like phenotypes. Interestingly, the proportion of ventricular-like cells was much higher in the Acta2-positive cardiomyocytes population than in control Α-MHC-expressing cardiomyocytes (75 % and 12 %, respectively). The findings of the present study provide a novel approach for the identification and enrichment of Acta2-positive cardiomyocytes, especially of the ventricular phenotype under in vitro conditions.

Putting the brakes on p53-driven apoptosis

Following genotoxic stress, cells activate a complex, kinase-based signaling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumor suppressor p53 lies at the heart of this DNA damage response. p53 mediates the transactivation of both cell cycle-regulating and pro-apoptotic clusters of target genes. However, it remains incompletely understood which signaling molecules dictate the choice between these two opposing p53-dependent cellular outcomes. Over recent years, numerous regulatory mechanisms impacting on the cellular outcome of p53 signaling have been described. However, no single dominant mechanism has thus far been identified to regulate the cellular choice between p53-driven apoptosis or senescence. The transcriptional regulator AATF has recently emerged as a novel factor impacting on the cellular outcome of the p53 response. Upon genotoxic stress, cytoplasmic pools of MRLC-bound AATF are phosphorylated through the p38MAPK/MK2 checkpoint kinase complex. This AATF phosphorylation results in the disruption of cytoplasmic MRLC3:AATF complexes followed by rapid nuclear localization of AATF. Once in the nucleus, AATF binds to the PUMA, BAX and BAK promoters to repress the DNA damage-induced expression of these pro-apoptotic p53 target genes. Depletion of AATF in tumor cells results in a dramatically enhanced response to DNA-damaging chemotherapeutics, both in vitro and in vivo. Furthermore, focal copy number gains at the AATF locus in neuroblastoma correlate with adverse prognosis and reduced overall survival in this typically p53-proficient malignancy. These data identify the p38/MK2/AATF signaling pathway as a critical repressor of p53-driven apoptosis in tumor cells and implicate this signaling cascade as a novel target for chemotherapy-sensitizing therapeutic efforts.

Optimization of the culturing conditions of human umbilical cord blood‐derived endothelial colony‐forming cells under xeno‐free conditions applying a transcriptomic approach

Establishment of fetal bovine serum (FBS)‐free cell culture conditions is essential for transplantation therapies. Blood‐derived endothelial colony‐forming cells (ECFCs) are potential candidates for regenerative medicine applications. ECFCs were isolated from term umbilical cord blood units and characterized by flow cytometry, capillary formation and responsiveness to cytokines. ECFCs were expanded under standard, FBS‐containing endothelial medium, or transferred to chemically defined endothelial media without FBS. Microarray expression profiling was applied to compare the transcriptome profiles in FBS‐containing versus FBS‐free culture. ECFC outgrowth in standard medium was successful in 92% of cord blood units. The karyotype of expanded ECFCs remained normal. Without FBS, ECFC initiation and expansion failed. Modest proliferation, changes in cell morphology and organization and cell death have been observed after passaging. Gene ontology analysis revealed a broad down‐regulation of genes involved in cell cycle progression and up‐regulation of genes involved in stress response and apoptosis. Interestingly, genes participating in lipid biosynthesis were markedly up‐regulated. Detection of several endothelial cell‐specific marker genes showed the maintenance of the endothelial cell characteristics during serum‐free culture. Although ECFCs maintain their endothelial characteristics during serum‐free culturing, they could not be expanded. Additional supply of FBS‐free media with lipid concentrates might increase the ECFC survival.