A Genome-wide Analysis of Human Pluripotent Stem Cell-Derived Endothelial Cells in 2D or 3D Culture

Summary: A defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for “3D-like” vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function. : Murphy, Schwartz and colleagues report a genome-wide analysis of biological function for endothelial cells and pericytes cultured using 2D or 3D formats. Global gene expression patterns are comparable for vascular cells cultured in synthetic hydrogels or Matrigel during vascular network formation, while cells cultured on tissue-culture polystyrene surfaces adopt a proliferative phenotype that correlates to increased FAK-ERK activity. Keywords: tissue engineering, MAPK, focal adhesion kinase, hydrogel, Matrigel, 3D culture, human pluripotent stem cells, endothelial cells, xeno-free, differentiatio

Comparative RNA-seq Analysis in the Unsequenced Axolotl: The Oncogene Burst Highlights Early Gene Expression in the Blastema

<div><p>The salamander has the remarkable ability to regenerate its limb after amputation. Cells at the site of amputation form a blastema and then proliferate and differentiate to regrow the limb. To better understand this process, we performed deep RNA sequencing of the blastema over a time course in the axolotl, a species whose genome has not been sequenced. Using a novel comparative approach to analyzing RNA-seq data, we characterized the transcriptional dynamics of the regenerating axolotl limb with respect to the human gene set. This approach involved de novo assembly of axolotl transcripts, RNA-seq transcript quantification without a reference genome, and transformation of abundances from axolotl contigs to human genes. We found a prominent burst in oncogene expression during the first day and blastemal/limb bud genes peaking at 7 to 14 days. In addition, we found that limb patterning genes, SALL genes, and genes involved in angiogenesis, wound healing, defense/immunity, and bone development are enriched during blastema formation and development. Finally, we identified a category of genes with no prior literature support for limb regeneration that are candidates for further evaluation based on their expression pattern during the regenerative process.</p> </div

Enrichment of oncogenes during the early time points.

<p>All upregulated DE genes at each time point (when compared to the zero hour control) were interrogated for being oncogenes by their presence in the Memorial Sloan Kettering Cancer Center Oncogene set (<a href="http://cbio.mskcc.org/CancerGenes/Select.action" target="_blank">http://cbio.mskcc.org/CancerGenes/Select.action</a>) and then performing a Fisher's exact test to assess significance. The enrichment of oncogenes during the first day is highly statistically significant (P-value<10⁻⁵). Late in the time course, oncogenes are not significantly enriched in the upregulated DE gene sets.</p

Bootstrapping of the sample clusters from <b>Figure 3</b>.

<p>To assess the uncertainty in hierarchical cluster analysis over samples, bootstrap resampling (10,000 iterations) was applied via the R package Pvclust <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002936#pcbi.1002936-Suzuki1" target="_blank">[26]</a>. The uncentered Pearson correlation is used as the distance metric with average linkage. The numbers above each edge show the probability of nodes below that edge occurring as a cluster in resampled trees, via ordinary bootstrap resampling (BP, green) or multiscale bootstrap resampling (AU, red). See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002936#s4" target="_blank">Materials and Methods</a> for details on bootstrapping.</p

Diagram of the blastemal time course experiment.

<p>RNA was harvested from tissue samples and subjected to deep sequencing on the Illumina GA II platform (see also <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002936#pcbi.1002936.s001" target="_blank">Figure S1</a>).</p

Expression of embryonic and adult stem cell genes in ES, iPS, FS, and blastemas.

<p>All samples are ratioed to the time zero axolotl juvenile time course control. While ES and iPS cells show upregulation of a variety of stem cell genes including the key ES transcription factors <i>POU5F1</i> and <i>NANOG</i>, the axolotl blastemas do not show upregulation of <i>POU5F1</i>, <i>SOX2</i>, and <i>NANOG</i>, but do show upregulation of adult stem cell markers (such as <i>SALL4</i> and <i>HMGA2</i>). Note the burst of oncogenes (<i>KLF4</i>, <i>MYC</i>) early in the time course and the upregulation of adult stem cell genes later in the time course.</p

Gene ontology (GO) analysis of the time course.

<p>GO enrichment of molecular function (MF) and biological process (BP) level 5 GO terms during the time course for all DE genes (relative to the zero hour control). The value displayed is the −log₁₀(GO term FDR). GO terms with an FDR<0.01 are shown. The GO terms were clustered by their enrichment patterns using the Pearson correlation as the distance measure with average linkage. During the first day enriched GO terms include GO terms representing immune response, chemotaxis, regulation of leukocytes, blood vessel development, and angiogenesis. In the middle of the time course (3 d–14 d), GO terms for tissue development, limb morphogenesis, bone development, and forebrain development are enriched. An ectoderm development GO term is enriched at 28 days. (Detailed GO enrichment information for upregulated axolotl genes is available at <a href="http://www.axolomics.org" target="_blank">www.axolomics.org</a>). (See also <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002936#pcbi.1002936.s010" target="_blank">Table S4</a> for the −log₁₀(GO term FDR) values.)</p

Expression of <i>SALL1</i>, <i>SALL3</i>, and <i>SALL4</i> in juvenile blastemas.

<p><i>SALL4</i> is upregulated earlier than <i>SALL1</i> or <i>SALL3</i>. “TPM” is the Transcripts Per Million expression measure.</p