The Transcriptomic Profiling Of Murine Embryonic Stem Cell-derived Mesodermal Cell Lineages And Functional Cardiomyocytes: An Insight Into Mesodermal Patterning

Summary The underlying basis of vertebrate mesodermal patterning by which the mesodermal cells become sequentially determined to more precisely defined cell fates during embryonic development remains largely unknown and is one of the classical problems in developmental biology. This demands an in-depth analysis of the endogenous signaling cascades and transcription factor networks. Till date, this fascinating puzzle is not yet solved since isolation of pure early stage mesodermal cells was not feasible due to practical difficulties and hence the functional genomics of the mesodermal cells has not yet been successful. Recently, embryonic stem (ES) cells have been proven to be a promising tool to study the early embryonic development in a more physiological context in vitro because of their versatility over the other conventional systems. Furthermore, ES cells may revolutionize medicine by providing an unlimited renewable source of cells capable of replacing or repairing tissues that have been damaged in all degenerative diseases. Using ES cell model in vitro, for the first time pure populations of T Brachyury and BMP-2 expressing mesodermal lineages and mesodermal derived pure and functional cardiomyocytes have been isolated and their entire transcriptomes were profiled using microarrays. To isolate the pure lineage specific cells, stable transgenic ES clones transfected with a targeting vector construct in which the promoter of lineage specific marker (T, BMP-2 or aMHC) drives the expression of both puromycin resistance and enhanced green fluorescence bicistronically with the help of an IRES element were generated. The EBs made from these clones were treated at the onset of the particular transcript expression with puromycin to enrich the respective lineages. Furthermore, analysis of the gene signatures specific to mesodermal cells and cardiomyocytes that defines their unique cellular and genetic identities contributing a major molecular insight into mesodermal patterning has been performed. In addition, a significant number of novel putative transcripts whose functions are unknown but expressed in specific lineages are enlisted. Few of these transcripts were functionally evaluated for their role in germ layer development and patterning. In addition, the purified populations of T Brachyury and BMP-2 mesodermal cells are capable of priming themselves to give rise to cardiomyocytes along with other mesodermal lineages. Interestingly, the BMP-2 positive cells contained predominantly neural crest stem cells (NCSCs) and their lineages namely smooth muscle cells, epithelial cells, melanocytes, and astrocytes along with cardiomyocytes. The transcriptome characterization of ES cell-derived aMHC cardiomyocytes clearly proves that these cells are exhibiting the properties more like the cardiomyocytes developed in in vivo conditions from the point of biological processes and hence are promising candidates for the future cardiac cell replacement therapy. In overall, the part of the work presented here will better contribute substantially to the lineage specific transcriptomic atlas which will shed light in understanding the complex embryonic developmental process

Overexpression of Map3k7 activates sinoatrial node-like differentiation in mouse ES-derived cardiomyocytes

In vivo, cardiomyocytes comprise a heterogeneous population of contractile cells defined by unique electrophysiologies, molecular markers and morphologies. The mechanisms directing myocardial cells to specific sub-lineages remain poorly understood. Here we report that overexpression of TGFβ-Activated Kinase (TAK1/Map3k7) in mouse embryonic stem (ES) cells faithfully directs myocardial differentiation of embryoid body (EB)-derived cardiac cells toward the sinoatrial node (SAN) lineage. Most cardiac cells in Map3k7-overexpressing EBs adopt markers, cellular morphologies, and electrophysiological behaviors characteristic of the SAN. These data, in addition to the fact that Map3k7 is upregulated in the sinus venous—the source of cells for the SAN—suggest that Map3k7 may be an endogenous regulator of the SAN fate

Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives

<p>Abstract</p> <p>Background</p> <p>Mouse embryonic stem (ES) cells remain pluripotent <it>in vitro </it>when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency.</p> <p>Results</p> <p>By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [<it>Pluri </it>genes], ii) LIF induced genes [<it>Lifind </it>genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of <it>Pluri </it>and <it>Lifind </it>genes, and revealed their differential modulation by <it>Oct4 </it>or <it>Nanog </it>master genes. Individual knock down of a selection of <it>Pluri </it>and <it>Lifind </it>genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed.</p> <p>Conclusion</p> <p>We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.</p

Global transcriptome analysis of murine embryonic stem cell-derived cardiomyocytes

Microarray analysis reveals that the specific pattern of gene expression in cardiomyocytes derived from embryonic stem cells reflects the biological, physiological and functional processes occurring in mature cardiomyocytes

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

Transcriptome analysis of BMP2+ cells in comparison to the undifferentiated BMP2 ES cells and the control population from 7-day old embryoid bodies led to the identification of 479 specifically upregulated and 193 downregulated transcripts

The FunGenES Database: A Genomics Resource for Mouse Embryonic Stem Cell Differentiation

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the “Functional Genomics in Embryonic Stem Cells” consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in “Expression Waves” and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells

eXtraembryonic ENdoderm (XEN) Stem Cells Produce Factors that Activate Heart Formation

Initial specification of cardiomyocytes in the mouse results from interactions between the extraembryonic anterior visceral endoderm (AVE) and the nascent mesoderm. However the mechanism by which AVE activates cardiogenesis is not well understood, and the identity of specific cardiogenic factors in the endoderm remains elusive. Most mammalian studies of the cardiogenic potential of the endoderm have relied on the use of cell lines that are similar to the heart-inducing AVE. These include the embryonal-carcinoma-derived cell lines, END2 and PYS2. The recent development of protocols to isolate eXtraembryonic ENdoderm (XEN) stem cells, representing the extraembryonic endoderm lineage, from blastocyst stage mouse embryos offers new tools for the genetic dissection of cardiogenesis.Here, we demonstrate that XEN cell-conditioned media (CM) enhances cardiogenesis during Embryoid Body (EB) differentiation of mouse embryonic stem (ES) cells in a manner comparable to PYS2-CM and END2-CM. Addition of CM from each of these three cell lines enhanced the percentage of EBs that formed beating areas, but ultimately, only XEN-CM and PYS2-CM increased the total number of cardiomyocytes that formed. Furthermore, our observations revealed that both contact-independent and contact-dependent factors are required to mediate the full cardiogenic potential of the endoderm. Finally, we used gene array comparison to identify factors in these cell lines that could mediate their cardiogenic potential.These studies represent the first step in the use of XEN cells as a molecular genetic tool to study cardiomyocyte differentiation. Not only are XEN cells functionally similar to the heart-inducing AVE, but also can be used for the genetic dissection of the cardiogenic potential of AVE, since they can be isolated from both wild type and mutant blastocysts. These studies further demonstrate the importance of both contact-dependent and contact-independent factors in cardiogenesis and identify potential heart-inducing proteins in the endoderm

A Comparative Analysis of Extra-Embryonic Endoderm Cell Lines

Prior to gastrulation in the mouse, all endodermal cells arise from the primitive endoderm of the blastocyst stage embryo. Primitive endoderm and its derivatives are generally referred to as extra-embryonic endoderm (ExEn) because the majority of these cells contribute to extra-embryonic lineages encompassing the visceral endoderm (VE) and the parietal endoderm (PE). During gastrulation, the definitive endoderm (DE) forms by ingression of cells from the epiblast. The DE comprises most of the cells of the gut and its accessory organs. Despite their different origins and fates, there is a surprising amount of overlap in marker expression between the ExEn and DE, making it difficult to distinguish between these cell types by marker analysis. This is significant for two main reasons. First, because endodermal organs, such as the liver and pancreas, play important physiological roles in adult animals, much experimental effort has been directed in recent years toward the establishment of protocols for the efficient derivation of endodermal cell types in vitro. Conversely, factors secreted by the VE play pivotal roles that cannot be attributed to the DE in early axis formation, heart formation and the patterning of the anterior nervous system. Thus, efforts in both of these areas have been hampered by a lack of markers that clearly distinguish between ExEn and DE. To further understand the ExEn we have undertaken a comparative analysis of three ExEn-like cell lines (END2, PYS2 and XEN). PYS2 cells are derived from embryonal carcinomas (EC) of 129 strain mice and have been characterized as parietal endoderm-like [1], END2 cells are derived from P19 ECs and described as visceral endoderm-like, while XEN cells are derived from blastocyst stage embryos and are described as primitive endoderm-like. Our analysis suggests that none of these cell lines represent a bona fide single in vivo lineage. Both PYS2 and XEN cells represent mixed populations expressing markers for several ExEn lineages. Conversely END2 cells, which were previously characterized as VE-like, fail to express many markers that are widely expressed in the VE, but instead express markers for only a subset of the VE, the anterior visceral endoderm. In addition END2 cells also express markers for the PE. We extended these observations with microarray analysis which was used to probe and refine previously published data sets of genes proposed to distinguish between DE and VE. Finally, genome-wide pathway analysis revealed that SMAD-independent TGFbeta signaling through a TAK1/p38/JNK or TAK1/NLK pathway may represent one mode of intracellular signaling shared by all three of these lines, and suggests that factors downstream of these pathways may mediate some functions of the ExEn. These studies represent the first step in the development of XEN cells as a powerful molecular genetic tool to study the endodermal signals that mediate the important developmental functions of the extra-embryonic endoderm. Our data refine our current knowledge of markers that distinguish various subtypes of endoderm. In addition, pathway analysis suggests that the ExEn may mediate some of its functions through a non-classical MAP Kinase signaling pathway downstream of TAK1

Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications

Induced pluripotent stem cell (iPSC)-based disease modelling and the cell replacement therapy approach have proven to be very powerful and instrumental in biomedical research and personalized regenerative medicine as evidenced in the past decade by unraveling novel pathological mechanisms of a multitude of monogenic diseases at the cellular level and the ongoing and emerging clinical trials with iPSC-derived cell products. iPSC-based disease modelling has sparked widespread enthusiasm and has presented an unprecedented opportunity in high throughput drug discovery platforms and safety pharmacology in association with three-dimensional multicellular organoids such as personalized organs-on-chips, gene/base editing, artificial intelligence and high throughput &ldquo;omics&rdquo; methodologies. This critical review summarizes the progress made in the past decade with the advent of iPSC discovery in biomedical applications and regenerative medicine with case examples and the current major challenges that need to be addressed to unleash the full potential of iPSCs in clinical settings and pharmacology for more effective and safer regenerative therapy

Current Challenges of iPSC-Based Disease Modeling and Therapeutic Implications

Induced pluripotent stem cell (iPSC)-based disease modelling and the cell replacement therapy approach have proven to be very powerful and instrumental in biomedical research and personalized regenerative medicine as evidenced in the past decade by unraveling novel pathological mechanisms of a multitude of monogenic diseases at the cellular level and the ongoing and emerging clinical trials with iPSC-derived cell products. iPSC-based disease modelling has sparked widespread enthusiasm and has presented an unprecedented opportunity in high throughput drug discovery platforms and safety pharmacology in association with three-dimensional multicellular organoids such as personalized organs-on-chips, gene/base editing, artificial intelligence and high throughput omics methodologies. This critical review summarizes the progress made in the past decade with the advent of iPSC discovery in biomedical applications and regenerative medicine with case examples and the current major challenges that need to be addressed to unleash the full potential of iPSCs in clinical settings and pharmacology for more effective and safer regenerative therapy