S. Doppler

Live fluorescent RNA-based detection of pluripotency gene expression in embryonic and induced pluripotent stem cells of different species.

The generation of induced pluripotent stem (iPS) cells has successfully been achieved in many species. However, the identification of truly reprogrammed iPS cells still remains laborious and the detection of pluripotency markers requires fixation of cells in most cases. Here, we report an approach with nanoparticles carrying Cy3‐labeled sense oligonucleotide reporter strands coupled to gold‐particles. These molecules are directly added to cultured cells without any manipulation and gene expression is evaluated microscopically after overnight incubation. To simultaneously detect gene expression in different species, probe sequences were chosen according to interspecies homology. With a common target‐specific probe we could successfully demonstrate expression of the GAPDH house‐keeping gene in somatic cells and expression of the pluripotency markers NANOG and GDF3 in embryonic stem cells and iPS cells of murine, human, and porcine origin. The population of target gene positive cells could be purified by fluorescence‐activated cell sorting. After lentiviral transduction of murine tail‐tip fibroblasts Nanog‐specific probes identified truly reprogrammed murine iPS cells in situ during development based on their Cy3‐fluorescence. The intensity of Nanog‐specific fluorescence correlated positively with an increased capacity of individual clones to differentiate into cells of all three germ layers. Our approach offers a universal tool to detect intracellular gene expression directly in live cells of any desired origin without the need for manipulation, thus allowing conservation of the genetic background of the target cell. Furthermore, it represents an easy, scalable method for efficient screening of pluripotency which is highly desirable during high‐throughput cell reprogramming and after genomic editing of pluripotent stem cells. Stem Cells 2015;33:392–402

Region and cell-type resolved quantitative proteomic map of the human heart.

The heart is a central human organ and its diseases are the leading cause of death worldwide, but an in-depth knowledge of the identity and quantity of its constituent proteins is still lacking. Here, we determine the healthy human heart proteome by measuring 16 anatomical regions and three major cardiac cell types by high-resolution mass spectrometry-based proteomics. From low microgram sample amounts, we quantify over 10,700 proteins in this high dynamic range tissue. We combine copy numbers per cell with protein organellar assignments to build a model of the heart proteome at the subcellular level. Analysis of cardiac fibroblasts identifies cellular receptors as potential cell surface markers. Application of our heart map to atrial fibrillation reveals individually distinct mitochondrial dysfunctions. The heart map is available at maxqb.biochem.mpg.de as a resource for future analyses of normal heart function and disease.The human heart is composed of distinct regions and cell types, but relatively little is known about their specific protein composition. Here, the authors present a region- and cell type-specific proteomic map of the healthy human heart, revealing functional differences and potential cell type markers.

Direct Reprogramming—The Future of Cardiac Regeneration?

Today, the only available curative therapy for end stage congestive heart failure (CHF) is heart transplantation. This therapeutic option is strongly limited by declining numbers of available donor hearts and by restricted long-term performance of the transplanted graft. The disastrous prognosis for CHF with its restricted therapeutic options has led scientists to develop different concepts of alternative regenerative treatment strategies including stem cell transplantation or stimulating cell proliferation of different cardiac cell types in situ. However, first clinical trials with overall inconsistent results were not encouraging, particularly in terms of functional outcome. Among other approaches, very promising ongoing pre-clinical research focuses on direct lineage conversion of scar fibroblasts into functional myocardium, termed “direct reprogramming” or “transdifferentiation.” This review seeks to summarize strategies for direct cardiac reprogramming including the application of different sets of transcription factors, microRNAs, and small molecules for an efficient generation of cardiomyogenic cells for regenerative purposes.

Mutational analysis of the human MESP1 gene in patients with congenital heart disease reveals a highly variable sequence in exon 1

MESP1 represents an essential transcription factor to guarantee coordinated cardiac development. The expression of MESP1 is thought to be the first sign that a cell has been committed to the cardiac lineage. We analyzed the coding sequence of MESP1 in 215 patients with congenital heart disease. Our results show that the sequence of exon 1 is highly variable with up to seven alterations in individual samples. Five base pair positions (c.157_G>C A53P, rs6496598; c.174_A>C P58P, rs28377352; c.182_T>G L61R, rs28368490; c.669_C>G F223L, rs2305440; c.687_T>G P229P, rs2305441) are particularly variable. In almost half of the samples a 12 base pair insertion after position 55 (c.165_166insGTGCCGAGCCCC P55insVPSP, rs71934166) coding for VPSP was detected which was strongly correlated with the appearance of further amino acid changes (c.157_G>C A53P, c.182_T>G L61R, c.669_C>G F223L). Two missense mutations (c.33_G>C E11D, rs190259690; c.528_A>T T176S) were detected in two patients but were absent in the controls. The assessment of the biological activity of altered MESP1 proteins in a luciferase reporter assay showed an enhanced activity of the c.33_G>C E11D mutation and a reduction of the insertion without an accompanying change of c.182_T>G L61R. The modified biological properties of mutated MESP1 proteins might be associated with the appearance of certain pathological phenotypes of congenital heart disease.

Cardiac fibroblasts: more than mechanical support

Fibroblasts are cells with a structural function, synthesizing components of the extracellular matrix. They are accordingly associated with various forms of connective tissue. During cardiac development fibroblasts originate from different sources. Most derive from the epicardium, some derive from the endocardium, and a small population derives from the neural crest. Cardiac fibroblasts have important functions during development, homeostasis, and disease. However, since fibroblasts are a very heterogeneous cell population no truly specific markers exist. Therefore, studying them in detail is difficult. Nevertheless, several lineage tracing models have been widely used. In this review, we describe the developmental origins of cardiac fibroblasts, comment on fibroblast markers and related lineage tracing approaches, and discuss the cardiac cell composition, which has recently been revised, especially in terms of non-myocyte cells.

MicroRNAs: pleiotropic players in congenital heart disease and regeneration

Congenital heart disease (CHD) is the leading cause of infant death, affecting approximately 4-14 live births per 1,000. Although surgical techniques and interventions have improved significantly, a large number of infants still face poor clinical outcomes. MicroRNAs (miRs) are known to coordinately regulate cardiac development and stimulate pathological processes in the heart, including fibrosis or hypertrophy and impair angiogenesis. Dysregulation of these regulators could therefore contribute (I) to the initial development of CHD and (II) at least partially to the observed clinical outcomes of many CHD patients by stimulating the aforementioned pathways. Thus, miRs may exhibit great potential as therapeutic targets in regenerative medicine. In this review we provide an overview of miR function and elucidate their role in selected CHDs, including hypoplastic left heart syndrome (HLHS), tetralogy of Fallot (TOF), ventricular septal defects (VSDs) and Holt-Oram syndrome (HOS). We then bridge this knowledge to the potential usefulness of miRs and/or their targets in therapeutic strategies for regenerative purposes in CHDs.

Tetralogy of Fallot and Hypoplastic Left Heart Syndrome - Complex Clinical Phenotypes Meet Complex Genetic Networks.

In many cases congenital heart disease (CHD) is represented by a complex phenotype and an array of several functional and morphological cardiac disorders. These malformations will be briefly summarized in the first part focusing on two severe CHD phenotypes, hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot (TOF). In most cases of CHD the genetic origin remains largely unknown, though the complexity of the clinical picture strongly argues against a dysregulation which can be attributed to a single candidate gene but rather suggests a multifaceted polygenetic origin with elaborate interactions. Consistent with this idea, genome-wide approaches using whole exome sequencing, comparative sequence analysis of multiplex families to identify de novo mutations and global technologies to identify single nucleotide polymorphisms, copy number variants, dysregulation of the transcriptome and epigenetic variations have been conducted to obtain information about genetic alterations and potential predispositions possibly linked to the occurrence of a CHD phenotype. In the second part of this review we will summarize and discuss the available literature on identified genetic alterations linked to TOF and HLHS.

Myeloid Zinc Finger 1 (Mzf1) Differentially Modulates Murine Cardiogenesis by Interacting with an Nkx2.5 Cardiac Enhancer

Vertebrate heart development is strictly regulated by temporal and spatial expression of growth and transcription factors (TFs). We analyzed nine TFs, selected by in silico analysis of an Nkx2.5 enhancer, for their ability to transactivate the respective enhancer element that drives, specifically, expression of genes in cardiac progenitor cells (CPCs). Mzf1 showed significant activity in reporter assays and bound directly to the Nkx2.5 cardiac enhancer (Nkx2.5 CE) during murine ES cell differentiation. While Mzf1 is established as a hematopoietic TF, its ability to regulate cardiogenesis is completely unknown. Mzf1 expression was significantly enriched in CPCs from in vitro differentiated ES cells and in mouse embryonic hearts. To examine the effect of Mzf1 overexpression on CPC formation, we generated a double transgenic, inducible, tetOMzf1-Nkx2.5 CE eGFP ES line. During in vitro differentiation an early and continuous Mzf1 overexpression inhibited CPC formation and cardiac gene expression. A late Mzf1 overexpression, coincident with a second physiological peak of Mzf1 expression, resulted in enhanced cardiogenesis. These findings implicate a novel, temporal-specific role of Mzf1 in embryonic heart development. Thereby we add another piece of puzzle in understanding the complex mechanisms of vertebrate cardiac development and progenitor cell differentiation. Consequently, this knowledge will be of critical importance to guide efficient cardiac regenerative strategies and to gain further insights into the molecular basis of congenital heart malformations.

Mammalian Heart Regeneration: The Race to the Finish Line

A long-standing goal of cardiovascular scientists is to repair damaged hearts after myocardial infarction by overcoming the limited regenerative capacity of the adult mammalian heart. Many new strategies are being actively investigated for generating functional cardiomyocytes or progenitor cells. The race is on for the first demonstration of durable therapeutic heart repair in human. The classical paradigm that the adult mammalian heart is a postmitotic, terminally differentiated organ has been actively debated. Data from 14C dating studies have demonstrated a limited self-renewal capacity of the adult mammalian heart that declines with age.1 These repair processes are, however, insufficient to compensate for extensive cell loss occurring after acute or chronic cardiac injury. Therefore, investigators in cardiovascular regenerative medicine have actively explored different approaches to replace the postinfarct scar tissue with contractile muscle cells. Despite these efforts, a central question for the field of cardiac regenerative medicine remains: what strategy will take us to the finish line in this race to achieve complete heart regeneration? In this Viewpoint article, we comment on recent experimental and clinical approaches to regenerate the failing heart and highlight the urgent need to clarify the biology of cardiomyocyte (CM) development, growth, and maturation. The mammalian heart harbors multiple cell types that may be targeted in regenerative strategies. Conceptually, the most direct approach is to stimulate pre-existing CMs to re-enter the cell cycle. Alternatively, resident cardiomyogenic stem/progenitor cell populations have been described to differentiate directly into CMs. Although there are advantages and disadvantages to each approach, it is clear that both strategies have demonstrated early promises that require further improvement and independent validation in nonrodent models. ### Neonatal Heart The regenerative capacity of the neonatal mammalian heart seems to be greater than that of adult heart because of the presence of a short window of CM proliferative competence.2 Immediately …

Small RNAs Make Big Impact in Cardiac Repair

During the past few decades, there has been enormous progress made in understanding and treating cardiovascular diseases. However, heart failure remains a progressive and debilitating condition with generally poor clinical outcomes and high socio-economic burden. The most common cause of heart failure is caused by loss of functional cardiomyocytes from myocardial infarction and subsequent fibrosis, leading to adverse remodeling, reduced contractile function, and hemodynamic compromise. Given the dire need for better heart failure treatment, investigators have actively explored strategies to improve cardiac function via numerous approaches, including cell transplantation, mechanical device support, or whole organ replacement.1 Although a detailed comparison of the merit of each of these approaches is beyond the scope of this article, one strategy that has captured tremendous interest in recent years is the use of highly potent transcription factors to reprogram cells into an alternative fate. The remarkable finding of Yamanaka and colleagues to revert a fully differentiated cell back to its most primitive state using a combination of transcript factors brought forth widespread optimism that a similar approach can be used successfully to reprogram any somatic cell into a different cell type.2–5 This subsequently led to several follow-on studies to directly reprogram fibroblasts into other cell lineages.6,7 In 2010, one such study by Ieda, Srivastava, and colleagues described the generation of cardiomyocyte-like cells (iCM) by overexpressing Gata4, Mef2c, and Tbx5 (GMT), transcription factors that have been shown to play important roles in cardiac development.8 Although this study reported the generation of 5% to 10% troponin T expressing cells in vitro, 2 follow up studies by Qian et al9 and Song et al10 report the ability of GMT or GMT+Hand1 to reprogram resident cardiac fibroblasts within the failing/fibrotic myocardium into functional …