Characterization and analysis of long non-coding rna (lncRNA) in <i>In Vitro</i>- and <i>Ex Vivo</i>-derived cardiac progenitor cells

<div><p>Recent advancements in cell-based therapies for the treatment of cardiovascular disease (CVD) show continuing promise for the use of transplanted stem and cardiac progenitor cells (CPCs) to promote cardiac restitution. However, a detailed understanding of the molecular mechanisms that control the development of these cells remains incomplete and is critical for optimizing their use in such therapy. Long non-coding (lnc) RNA has recently emerged as a crucial class of regulatory molecules involved in directing a variety of critical biological processes including development, homeostasis and disease. As such, a rising body of evidence suggests that they also play key regulatory roles in CPC development, though many questions remain regarding the expression landscape and specific identity of lncRNA involved in this process. To address this, we performed whole transcriptome sequencing of two murine CPC populations–Nkx2-5 EmGFP reporter-sorted embryonic stem (ES) cell-derived and <i>ex vivo</i>, cardiosphere-derived–in an effort to characterize their lncRNA profiles and potentially identify novel CPC regulators. The resulting sequencing data revealed an enrichment in both CPC populations for a panel of previously-identified lncRNA genes associated with cardiac differentiation. Additionally, a total of 1,678 differentially expressed and as-of-yet unannotated, putative lncRNA genes were found to be enriched for in the two CPC populations relative to undifferentiated ES cells.</p></div

Gene expression dynamics for pluripotency-, mesoderm- and cardiac lineage-associated transcription factors in cardiac-differentiated Nkx2.5 EmGFP ES cells.

<p><b>A)</b> pluripotency markers Nanog, Oct4 and Sox2, <b>B)</b> mesodermal marker Brachyury T and early cardiac lineage transcription factors Mesp1, Nkx2.5, Tbx5 and Isl1, <b>C)</b> cardiac-specific contractile proteins Myh6 and Tnnt2 (*p<0.05 vs. d0 undifferentiated; n = 3).</p

RNA-Seq data for d8 and CLK CPCs confirms enrichment for protein-coding and lncRNA genes associated with CPCs as well as the distribution of differentially-expressed annotated and unannotated genes.

<p><b>A)</b> RNA-seq-derived RPKM values for pluripotency and cardiac-related genes as proportionally represented by d8/CLK CPCs and undifferentiated ES cells, <b>B)</b> distribution of RPKM values for d8/CLK CPCs and undifferentiated ES cells with respect to known cardiac-related lncRNAs, <b>C)</b> graphical summary of d8/CLK enrichment for panel of lncRNA genes, previously identified by Wamstead et al, in cardiac-differentiated ES cells, <b>D)</b> Biaxial Venn plot showing distribution of differentially-expressed annotated and unannotated genes in d8/CLK CPCs relative to undifferentiated ES cells, highlighting transcripts that are commonly up- and down-regulated.</p

Sashimi read density plots show unannotated, putative lncRNAs common to d8/CLK CPCs that are differentially expressed relative to undifferentiated ES cells and map adjacent to cardiac-related protein-coding genes.

<p><b>A)</b> 247nt transcript commonly upregulated in d8/CLK CPCs, approximately 1Kb upstream of Adamts6, B) 2,024nt transcript upregulated in d8/CLK CPCs, immediately upstream of Ctdspl, C) 1,378nt transcript downregulated in d8/CLK CPCs, upstream of Ccnb1ip1.</p

Schematic overview and FACS analysis of <i>in vitro</i>, murine Nkx2.5 EmGFP ES cell-based cardiac differentiation.

<p><b>A)</b> schematic representation of homologously recombineered, 2A self-cleaving EmGFP reporter inserted immediately downstream of the endogenous Nkx2.5 promoter and immediately upstream of the Nkx2.5 gene in mouse ES cells, <b>B)</b> overview of gene expression trends for <i>in vitro</i> cardiac-directed differentiation, sorting and purification of Nkx2.5 EmGFP reporter ES cells, <b>C)</b> FACS analysis of Nkx2.5 EmGFP ES cells at d6, d8 and d10 of cardiac differentiation, <b>D)</b> EmGFP gene expression kinetics for non-sorted Nkx2.5 EmGFP ES cells throughout cardiac differentiation (* p<0.05, n = 3).</p

Experimental approach for the identification of protein–protein interactions using ODM BLAST

<p><b>Copyright information:</b></p><p>Taken from "Oracle Database 10: a platform for BLAST search and Regular Expression pattern matching in life sciences"</p><p>Nucleic Acids Research 2004 ;33(Database Issue):D675-D679.</p><p>Published online 17 Dec 2004 </p><p>PMCID:PMC540068.</p><p>Copyright © 2005 Oxford University Press</p

A sample of the protein–protein interaction results gained from ODM BLAST analysis

<p><b>Copyright information:</b></p><p>Taken from "Oracle Database 10: a platform for BLAST search and Regular Expression pattern matching in life sciences"</p><p>Nucleic Acids Research 2004 ;33(Database Issue):D675-D679.</p><p>Published online 17 Dec 2004 </p><p>PMCID:PMC540068.</p><p>Copyright © 2005 Oxford University Press</p

qRT-PCR array validation of the randomly selected miRNAs.

<p>Plot graph shows correlation between expression levels of miRNAs measured by array analysis and expression levels measured by individual qRT-PCR. −ΔΔCt method was applied for this analysis.</p

List of selected most significant up and down-regulated expression miRNAs in invasive candidiasis.

<p>List of selected most significant up and down-regulated expression miRNAs in invasive candidiasis.</p

Aberrant expression of hsa-miR-16-1 and hsa-miR-155 (the most significant genes based on fold change and p-values).

<p>hsa-miR-16-1 is up-regulated while hsa-miR-155 miRNA is down-regulated in individuals infected with invasive candidiasis as compared to normal expression levels (control).</p