Leng Han

Patient-Specific Induced Pluripotent Stem Cells as a Model for Familial Dilated Cardiomyopathy

Human induced pluripotent stem cells generated from patients with familial dilated cardiomyopathy model cardiovascular disease in these patients. iPSCs Make the Heart Beat Faster Mutations in genes expressed in the heart can cause dilated cardiomyopathy (DCM), a form of heart disease in which a weakened and enlarged heart is unable to pump sufficient blood for the body’s needs. DCM can lead to progressive heart failure that eventually requires heart transplantation. This disease has been challenging to study because cardiomyocytes from the hearts of DCM patients are difficult to obtain and do not survive long. Mouse models of DCM are established and have provided important clues about the disease mechanisms for DCM. However, the mouse heart is very different in physiology compared to the human heart; for example, the mouse heart rate is 10 times faster than that of human. In a new study, Sun et al. generated induced pluripotent stem cells (iPSCs) from skin cells of patients in a family with inherited DCM. This family carries a deleterious mutation in TNNT2, a gene that is expressed specifically in the heart and regulates cardiomyocyte contraction. Using iPSCs, the authors generated a large number of individual-specific cardiomyocytes carrying the specific TNNT2 mutation and analyzed their functional properties. Compared to cardiomyocytes derived from iPSCs of healthy controls in the same family, cardiomyocytes derived from iPSCs of DCM patients exhibited an increased heterogeneous myofilament organization, susceptibility to stress, compromised ability to regulate calcium flux, and decreased contraction force. These results suggest that the mutation in TNNT2 causes abnormalities in the cardiomyocytes and contributes to the development of DCM disease. Using these DCM iPSC–derived cardiomyocytes, the researchers also showed that several current treatments that clinically benefit DCM disease improved DCM cardiomyocyte function in culture. The current study shows that human iPSC-derived cardiomyocytes could provide an important platform to investigate the specific disease mechanisms of DCM as well as other inherited cardiovascular disorders and for screening new drugs for cardiovascular disease. Characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure, dilated cardiomyopathy (DCM) is the most common form of cardiomyopathy in patients. DCM is the most common diagnosis leading to heart transplantation and places a significant burden on healthcare worldwide. The advent of induced pluripotent stem cells (iPSCs) offers an exceptional opportunity for creating disease-specific cellular models, investigating underlying mechanisms, and optimizing therapy. Here, we generated cardiomyocytes from iPSCs derived from patients in a DCM family carrying a point mutation (R173W) in the gene encoding sarcomeric protein cardiac troponin T. Compared to control healthy individuals in the same family cohort, cardiomyocytes derived from iPSCs from DCM patients exhibited altered regulation of calcium ion (Ca2+), decreased contractility, and abnormal distribution of sarcomeric α-actinin. When stimulated with a β-adrenergic agonist, DCM iPSC–derived cardiomyocytes showed characteristics of cellular stress such as reduced beating rates, compromised contraction, and a greater number of cells with abnormal sarcomeric α-actinin distribution. Treatment with β-adrenergic blockers or overexpression of sarcoplasmic reticulum Ca2+ adenosine triphosphatase (Serca2a) improved the function of iPSC-derived cardiomyocytes from DCM patients. Thus, iPSC-derived cardiomyocytes from DCM patients recapitulate to some extent the morphological and functional phenotypes of DCM and may serve as a useful platform for exploring disease mechanisms and for drug screening.

Assessing the clinical utility of cancer genomic and proteomic data across tumor types

Molecular profiling of tumors promises to advance the clinical management of cancer, but the benefits of integrating molecular data with traditional clinical variables have not been systematically studied. Here we retrospectively predict patient survival using diverse molecular data (somatic copy-number alteration, DNA methylation and mRNA, microRNA and protein expression) from 953 samples of four cancer types from The Cancer Genome Atlas project. We find that incorporating molecular data with clinical variables yields statistically significantly improved predictions (FDR < 0.05) for three cancers but those quantitative gains were limited (2.2–23.9%). Additional analyses revealed little predictive power across tumor types except for one case. In clinically relevant genes, we identified 10,281 somatic alterations across 12 cancer types in 2,928 of 3,277 patients (89.4%), many of which would not be revealed in single-tumor analyses. Our study provides a starting point and resources, including an open-access model evaluation platform, for building reliable prognostic and therapeutic strategies that incorporate molecular data.

The Genomic Landscape and Clinical Relevance of A-to-I RNA Editing in Human Cancers

Adenosine-to-inosine (A-to-I) RNA editing is a widespread post-transcriptional mechanism, but its genomic landscape and clinical relevance in cancer have not been investigated systematically. We characterized the global A-to-I RNA editing profiles of 6,236 patient samples of 17 cancer types from The Cancer Genome Atlas and revealed a striking diversity of altered RNA-editing patterns in tumors relative to normal tissues. We identified an appreciable number of clinically relevant editing events, many of which are in noncoding regions. We experimentally demonstrated the effects of several cross-tumor nonsynonymous RNA editing events on cell viability and provide the evidence that RNA editing could selectively affect drug sensitivity. These results highlight RNA editing as an exciting theme for investigating cancer mechanisms, biomarkers, and treatments.

Gene co-expression network analysis reveals common system-level properties of prognostic genes across cancer types

Prognostic genes are key molecules informative for cancer prognosis and treatment. Previous studies have focused on the properties of individual prognostic genes, but have lacked a global view of their system-level properties. Here we examined their properties in gene co-expression networks for four cancer types using data from The Cancer Genome Atlas. We found that prognostic mRNA genes tend not to be hub genes (genes with an extremely high connectivity), and this pattern is unique to the corresponding cancer-type specific network. In contrast, the prognostic genes are enriched in modules (., a group of highly interconnected genes), especially in module genes conserved across different cancer co-expression networks. The target genes of prognostic miRNA genes show similar patterns. We identified the modules enriched in various prognostic genes, some of which show cross-tumor conservation. Given the cancer types surveyed, our study presents a view of emergent properties of prognostic genes.

MicroRNA‐302 Increases Reprogramming Efficiency via Repression of NR2F2

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression through translational inhibition and RNA decay and have been implicated in the regulation of cellular differentiation, proliferation, angiogenesis, and apoptosis. In this study, we analyzed global miRNA and mRNA microarrays to predict novel miRNA‐mRNA interactions in human embryonic stem cells and induced pluripotent stem cells (iPSCs). In particular, we demonstrate a regulatory feedback loop between the miR‐302 cluster and two transcription factors, NR2F2 and OCT4. Our data show high expression of miR‐302 and OCT4 in pluripotent cells, while NR2F2 is expressed exclusively in differentiated cells. Target analysis predicts that NR2F2 is a direct target of miR‐302, which we experimentally confirm by reporter luciferase assays and real‐time polymerase chain reaction. We also demonstrate that NR2F2 directly inhibits the activity of the OCT4 promoter and thus diminishes the positive feedback loop between OCT4 and miR‐302. Importantly, higher reprogramming efficiencies were obtained when we reprogrammed human adipose‐derived stem cells into iPSCs using four factors (KLF4, C‐MYC, OCT4, and SOX2) plus miR‐302 (this reprogramming cocktail is hereafter referred to as “KMOS3”) when compared to using four factors (“KMOS”). Furthermore, shRNA knockdown of NR2F2 mimics the over‐expression of miR‐302 by also enhancing reprogramming efficiency. Interestingly, we were unable to generate iPSCs from miR‐302a/b/c/d alone, which is in contrast to previous publications that have reported that miR‐302 by itself can reprogram human skin cancer cells and human hair follicle cells. Taken together, these findings demonstrate that miR‐302 inhibits NR2F2 and promotes pluripotency through indirect positive regulation of OCT4. This feedback loop represents an important new mechanism for understanding and inducing pluripotency in somatic cells. STEM CELLS2013;31:259–268

Central Role of Mitofusin 2 in Autophagosome-Lysosome Fusion in Cardiomyocytes

Background: Mitofusin 2 is a mitochondrial outer membrane protein with multiple functions. Results: Mitofusin 2 deficiency in the heart causes autophagosomes to accumulate and leads to cardiac dysfunction. Conclusion: Mitofusin 2 serves as an adaptor protein to mediate the fusion of autophagosomes with lysosomes in the heart. Significance: Mitofusin 2 has a novel and essential role in cardiac autophagic regulation. In the heart, autophagy has been implicated in cardioprotection and ischemia-reperfusion tolerance, and the dysregulation of autophagy is associated with the development of heart failure. Mitochondrial dynamic proteins are profoundly involved in autophagic processes, especially the initiation and formation of autophagosomes, but it is not clear whether they play any role in cardiac autophagy. We previously reported that mitofusin 2 (MFN2), a mitochondrial outer membrane protein, serves as a major determinant of cardiomyocyte apoptosis mediated by oxidative stress. Here, we reveal a novel and essential role of MFN2 in mediating cardiac autophagy. We found that specific deletion of MFN2 in cardiomyocytes caused extensive accumulation of autophagosomes. In particular, the fusion of autophagosomes with lysosomes, a critical step in autophagic degradation, was markedly retarded without altering the formation of autophagosomes and lysosomes in response to ischemia-reperfusion stress. Importantly, MFN2 co-immunoprecipitated with RAB7 in the heart, and starvation further increased it. Knockdown of MFN2 by shRNA prevented, whereas re-expression of MFN2 restored, the autophagosome-lysosome fusion in neonatal cardiomyocytes. Hearts from cardiac-specific MFN2 knock-out mice had abnormal mitochondrial and cellular metabolism and were vulnerable to ischemia-reperfusion challenge. Our study defined a novel and essential role of MFN2 in the cardiac autophagic process by mediating the maturation of autophagy at the phase of autophagosome-lysosome fusion; deficiency of MFN2 caused multiple molecular and functional defects that undermined cardiac reserve and gradually led to cardiac vulnerability and dysfunction.

Dissecting the Molecular Relationship Among Various Cardiogenic Progenitor Cells

Rationale: Multiple progenitors derived from the heart and bone marrow (BM) have been used for cardiac repair. Despite this, not much is known about the molecular identity and relationship among these progenitors. To develop a robust stem cell therapy for the heart, it is critical to understand the molecular identity of the multiple cardiogenic progenitor cells. Objective: This study is the first report of high-throughput transcriptional profiling of cardiogenic progenitor cells carried out on an identical platform. Method and Results: Microarray-based transcriptional profiling was carried out for 3 cardiac (ckit+, Sca1+, and side population) and 2 BM (ckit+ and mesenchymal stem cell) progenitors, obtained from age- and sex-matched wild-type C57BL/6 mice. Analysis indicated that cardiac-derived ckit+ population was very distinct from Sca1+ and side population cells in the downregulation of genes encoding for cell–cell and cell–matrix adhesion proteins, and in the upregulation of developmental genes. Significant enrichment of transcripts involved in DNA replication and repair was observed in BM-derived progenitors. The BM ckit+ cells seemed to have the least correlation with the other progenitors, with enrichment of immature neutrophil–specific molecules. Conclusions: Our study indicates that cardiac ckit+ cells represent the most primitive population in the rodent heart. Primitive cells of cardiac versus BM origin differ significantly with respect to stemness and cardiac lineage–specific genes, and molecules involved in DNA replication and repair. The detailed molecular profile of progenitors reported here will serve as a useful reference to determine the molecular identity of progenitors used in future preclinical and clinical studies.

LncRNA NBR2 engages a metabolic checkpoint by regulating AMPK under energy stress

Long non-coding RNAs (lncRNAs) have emerged as critical regulators in various cellular processes. However, the potential involvement of lncRNAs in kinase signalling remains largely unknown. AMP-activated protein kinase (AMPK) acts as a critical sensor of cellular energy status. Here we show that the lncRNA NBR2 (neighbour of BRCA1 gene 2) is induced by the LKB1–AMPK pathway under energy stress. On energy stress, NBR2 in turn interacts with AMPK and promotes AMPK kinase activity, thus forming a feed-forward loop to potentiate AMPK activation during energy stress. Depletion of NBR2 attenuates energy-stress-induced AMPK activation, resulting in unchecked cell cycling, altered apoptosis/autophagy response, and increased tumour development in vivo. NBR2 is downregulated and its low expression correlates with poor clinical outcomes in some human cancers. Together, the results of our study uncover a mechanism coupling lncRNAs with metabolic stress response, and provides a broad framework to understand further the regulation of kinase signalling by lncRNAs.

INO80 Facilitates Pluripotency Gene Activation in Embryonic Stem Cell Self-Renewal, Reprogramming, and Blastocyst Development

The master transcription factors play integral roles in the pluripotency transcription circuitry of embryonic stem cells (ESCs). How they selectively activate expression of the pluripotency network while simultaneously repressing genes involved in differentiation is not fully understood. Here, we define a requirement for the INO80 complex, a SWI/SNF family chromatin remodeler, in ESC self-renewal, somatic cell reprogramming, and blastocyst development. We show that Ino80, the chromatin remodeling ATPase, co-occupies pluripotency gene promoters with the master transcription factors, and its occupancy is dependent on OCT4 and WDR5. At the pluripotency genes, Ino80 maintains an open chromatin architecture and licenses recruitment of Mediator and RNA polymerase II for gene activation. Our data reveal an essential role for INO80 in the expression of the pluripotency network and illustrate the coordination among chromatin remodeler, transcription factor, and histone-modifying enzyme in the regulation of the pluripotent state.

CpG island density and its correlations with genomic features in mammalian genomes

BackgroundCpG islands, which are clusters of CpG dinucleotides in GC-rich regions, are considered gene markers and represent an important feature of mammalian genomes. Previous studies of CpG islands have largely been on specific loci or within one genome. To date, there seems to be no comparative analysis of CpG islands and their density at the DNA sequence level among mammalian genomes and of their correlations with other genome features.ResultsIn this study, we performed a systematic analysis of CpG islands in ten mammalian genomes. We found that both the number of CpG islands and their density vary greatly among genomes, though many of these genomes encode similar numbers of genes. We observed significant correlations between CpG island density and genomic features such as number of chromosomes, chromosome size, and recombination rate. We also observed a trend of higher CpG island density in telomeric regions. Furthermore, we evaluated the performance of three computational algorithms for CpG island identifications. Finally, we compared our observations in mammals to other non-mammal vertebrates.ConclusionOur study revealed that CpG islands vary greatly among mammalian genomes. Some factors such as recombination rate and chromosome size might have influenced the evolution of CpG islands in the course of mammalian evolution. Our results suggest a scenario in which an increase in chromosome number increases the rate of recombination, which in turn elevates GC content to help prevent loss of CpG islands and maintain their density. These findings should be useful for studying mammalian genomes, the role of CpG islands in gene function, and molecular evolution.