Aitor Aguirre

Conversion of human fibroblasts to angioblast-like progenitor cells

Lineage conversion of one somatic cell type to another is an attractive approach for generating specific human cell types. Lineage conversion can be direct, in the absence of proliferation and multipotent progenitor generation, or indirect, by the generation of expandable multipotent progenitor states. We report the development of a reprogramming methodology in which cells transition through a plastic intermediate state, induced by brief exposure to reprogramming factors, followed by differentiation. We use this approach to convert human fibroblasts to mesodermal progenitor cells, including by non-integrative approaches. These progenitor cells demonstrated bipotent differentiation potential and could generate endothelial and smooth muscle lineages. Differentiated endothelial cells exhibited neo-angiogenesis and anastomosis in vivo. This methodology for indirect lineage conversion to angioblast-like cells adds to the armamentarium of reprogramming approaches aimed at the study and treatment of ischemic pathologies.

In Vivo Activation of a Conserved MicroRNA Program Induces Mammalian Heart Regeneration

Heart failure is a leading cause of mortality and morbidity in the developed world, partly because mammals lack the ability to regenerate heart tissue. Whether this is due to evolutionary loss of regenerative mechanisms present in other organisms or to an inability to activate such mechanisms is currently unclear. Here we decipher mechanisms underlying heart regeneration in adult zebrafish and show that the molecular regulators of this response are conserved in mammals. We identified miR-99/100 and Let-7a/c and their protein targets smarca5 and fntb as critical regulators of cardiomyocyte dedifferentiation and heart regeneration in zebrafish. Although human and murine adult cardiomyocytes fail to elicit an endogenous regenerative response after myocardial infarction, we show that in vivo manipulation of this molecular machinery in mice results in cardiomyocyte dedifferentiation and improved heart functionality after injury. These data provide a proof of concept for identifying and activating conserved molecular programs to regenerate the damaged heart.

Identification of Novel Long Noncoding RNAs Underlying Vertebrate Cardiovascular Development

Background— Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators with important functions in development and disease. Here, we sought to identify and functionally characterize novel lncRNAs critical for vertebrate development. Methods and Results— By relying on human pluripotent stem cell differentiation models, we investigated lncRNAs differentially regulated at key steps during human cardiovascular development with a special focus on vascular endothelial cells. RNA sequencing led to the generation of large data sets that serve as a gene expression roadmap highlighting gene expression changes during human pluripotent cell differentiation. Stage-specific analyses led to the identification of 3 previously uncharacterized lncRNAs, TERMINATOR, ALIEN, and PUNISHER, specifically expressed in undifferentiated pluripotent stem cells, cardiovascular progenitors, and differentiated endothelial cells, respectively. Functional characterization, including localization studies, dynamic expression analyses, epigenetic modification monitoring, and knockdown experiments in lower vertebrates, as well as murine embryos and human cells, confirmed a critical role for each lncRNA specific for each analyzed developmental stage. Conclusions— We have identified and functionally characterized 3 novel lncRNAs involved in vertebrate and human cardiovascular development, and we provide a comprehensive transcriptomic roadmap that sheds new light on the molecular mechanisms underlying human embryonic development, mesodermal commitment, and cardiovascular specification.

Reprogramming toward Heart Regeneration: Stem Cells and Beyond

Finding a cure for cardiovascular disease remains a major unmet medical need. Recent investigations have started to unveil the mechanisms of mammalian heart regeneration. The study of the regenerative mechanisms in lower vertebrate and mammalian animal models has provided clues for the experimental activation of proregenerative responses in the heart. In parallel, the use of endogenous adult stem cell populations alongside the recent application of reprogramming technologies has created major expectations for the development of therapies targeting heart disease. Together, these new approaches are bringing us closer to more successful strategies for the treatment of heart disease.

iPSCORE: A Resource of 222 iPSC Lines Enabling Functional Characterization of Genetic Variation across a Variety of Cell Types.

Summary Large-scale collections of induced pluripotent stem cells (iPSCs) could serve as powerful model systems for examining how genetic variation affects biology and disease. Here we describe the iPSCORE resource: a collection of systematically derived and characterized iPSC lines from 222 ethnically diverse individuals that allows for both familial and association-based genetic studies. iPSCORE lines are pluripotent with high genomic integrity (no or low numbers of somatic copy-number variants) as determined using high-throughput RNA-sequencing and genotyping arrays, respectively. Using iPSCs from a family of individuals, we show that iPSC-derived cardiomyocytes demonstrate gene expression patterns that cluster by genetic background, and can be used to examine variants associated with physiological and disease phenotypes. The iPSCORE collection contains representative individuals for risk and non-risk alleles for 95% of SNPs associated with human phenotypes through genome-wide association studies. Our study demonstrates the utility of iPSCORE for examining how genetic variants influence molecular and physiological traits in iPSCs and derived cell lines.

Establishment of human iPSC-based models for the study and targeting of glioma initiating cells

Glioma tumour-initiating cells (GTICs) can originate upon the transformation of neural progenitor cells (NPCs). Studies on GTICs have focused on primary tumours from which GTICs could be isolated and the use of human embryonic material. Recently, the somatic genomic landscape of human gliomas has been reported. RTK (receptor tyrosine kinase) and p53 signalling were found dysregulated in ∼90% and 86% of all primary tumours analysed, respectively. Here we report on the use of human-induced pluripotent stem cells (hiPSCs) for modelling gliomagenesis. Dysregulation of RTK and p53 signalling in hiPSC-derived NPCs (iNPCs) recapitulates GTIC properties in vitro. In vivo transplantation of transformed iNPCs leads to highly aggressive tumours containing undifferentiated stem cells and their differentiated derivatives. Metabolic modulation compromises GTIC viability. Last, screening of 101 anti-cancer compounds identifies three molecules specifically targeting transformed iNPCs and primary GTICs. Together, our results highlight the potential of hiPSCs for studying human tumourigenesis.

A lncRNA Perspective into (Re)Building the Heart

Our conception of the human genome, long focused on the 2% that codes for proteins, has profoundly changed since its first draft assembly in 2001. Since then, an unanticipatedly expansive functionality and convolution has been attributed to the majority of the genome that is transcribed in a cell-type/context-specific manner into transcripts with no apparent protein coding ability. While the majority of these transcripts, currently annotated as long non-coding RNAs (lncRNAs), are functionally uncharacterized, their prominent role in embryonic development and tissue homeostasis, especially in the context of the heart, is emerging. In this review, we summarize and discuss the latest advances in understanding the relevance of lncRNAs in (re)building the heart.

Comparison of Non-human Primate versus Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Treatment of Myocardial Infarction

Summary Non-human primates (NHPs) can serve as a human-like model to study cell therapy using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). However, whether the efficacy of NHP and human iPSC-CMs is mechanistically similar remains unknown. To examine this, RNU rats received intramyocardial injection of 1 × 107 NHP or human iPSC-CMs or the same number of respective fibroblasts or PBS control (n = 9–14/group) at 4 days after 60-min coronary artery occlusion-reperfusion. Cardiac function and left ventricular remodeling were similarly improved in both iPSC-CM-treated groups. To mimic the ischemic environment in the infarcted heart, both cultured NHP and human iPSC-CMs underwent 24-hr hypoxia in vitro. Both cells and media were collected, and similarities in transcriptomic as well as metabolomic profiles were noted between both groups. In conclusion, both NHP and human iPSC-CMs confer similar cardioprotection in a rodent myocardial infarction model through relatively similar mechanisms via promotion of cell survival, angiogenesis, and inhibition of hypertrophy and fibrosis.

Heart Regeneration A Tale of Cell Reprogramming

Is reprogramming a physiological repair mechanism occurring during cardiac regeneration? Heart failure is one of the main health problems worldwide, and the urgent medical needs relating to it have positioned cardiovascular research as one of the most actively evolving fields in regenerative medicine. Many different strategies aiming to revert, palliate, or ameliorate the deleterious effects of heart failure have been intensively pursued, and excellent reviews on the topic exist.1–3 Briefly, approaches used to tackle cardiac repair have been focused mainly on the use of stem cells to replenish lost muscle mass (cell transplantation or mobilization of resident cardiac stem cells), reduction of cardiomyocyte hypertrophy or prevention of fibrosis, promotion of angiogenesis, and, most recently, in vivo reprogramming strategies.4–6 Although these significant advances are contributing to the development of more efficient therapies, successful and efficient heart repair strategies are by and large still lacking. Are there alternative approaches beside those mentioned above? A possible line of attack is to look at how nature deals with this issue. Regenerative animal models are able to regenerate the heart completely.7 They do so by preventing scar formation, replenishing lost muscle tissue, and eliciting a controlled proangiogenic response. All these processes lead to a complete repair of the injured heart. Whereas it is well known that these processes occur naturally only in lower vertebrates, it has been a long established dogma that the mammalian heart is not able to regenerate. However, although the adult mammalian heart is not able to regenerate after massive myocardial damage, it can activate a healing process resembling that of regenerative organisms.8 Although the numbers of newly generated cardiomyocytes are insufficient for healing of the heart, it is clear that endogenous repair mechanisms are present in the mammalian heart, thus providing a potential …