Comparative RNA-seq analysis in the unsequenced axolotl: the oncogene burst highlights early gene expression in the blastema.

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.

Co-culture with mouse embryonic fibroblasts improves maintenance of metabolic function of human small hepatocyte progenitor cells.

Derivation and culture of small hepatocyte progenitor cells (SHPCs) capable of proliferating in vitro has been described in rodents and recently in humans. These cells are capable of engrafting in injured livers, however, they display de-differentiated morphology and reduced xenobiotic metabolism activity in culture over passages. Here we report that SHPCs derived from adult primary human hepatocytes (PHHs) and cultured on mouse embryonic fibroblasts (MEFs) not only display differentiated morphology and exhibit gene expression profiles similar to adult PHHs, but importantly, they retain their phenotype over several passages. Further, unlike previous reports, where extensive manipulations of culture conditions are required to convert SHPCs to metabolically functional hepatocytes, SHPCs in our co-culture system maintain expression of xenobiotic metabolism-associated genes. We show that SHPCs in co-culture are able to perform xenobiotic metabolism at rates equal to their parent PHHs as evidenced by the metabolism of acetaminophen to all of its major metabolites. In summary, we present an improved co-culture system that allows generation of SHPCs from adult PHHs that maintain their differentiated phenotype over multiple passages. Our findings would be useful for expansion of limited PHHs for use in studies of drug metabolism and toxicity testing.

3-D culture and endothelial cells improve maturity of human pluripotent stem cell-derived hepatocytes.

Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes (iHEP) offer an attractive alternative to primary human hepatocytes (PHH) for drug toxicity studies, as PHHs are limited in supply, vary in their metabolic activity between donors, and rapidly lose their functionality in vitro. However, one of the major drawbacks with iHEP cells in drug safety studies is their decreased phenotypic maturity, with lower liver specific enzyme activity compared with that of PHH. Here we evaluated the effects of 3D culture and non-parenchymal cells on the maturation of iHEPs. We describe a serum-free, chemically defined 3D in vitro model using iHEP cells, which is compatible with automation and conventional assay plates. The iHEP cells cultured in this model form polarized aggregates with functional bile canaliculi and strongly increased expression of albumin, urea and genes encoding phase I and II drug metabolism enzymes and bile transporters. Cytochrome P450-mediated metabolism is significantly higher in 3D iHEP aggregates compared to 2D iHEP culture. Furthermore, addition of human liver sinusoidal endothelial cells (sECs) and iPS-derived endothelial cells (iECs) improved mature hepatocyte function and CYP450 enzyme activity. Also, ECs formed endothelial networks within the hepatic 3D cultures, mimicking aspects of an in vivo architecture. Collectively, these results suggest that the iHEP/EC aggregates described here may have the potential to be used for many applications, including as an in vitro model to study liver diseases associated with sinusoidal endothelial cells. STATEMENT OF SIGNIFICANCE: iPS-derived hepatocytes provide an inexhaustible source of cells for drug screening, toxicology studies and cell-based therapies, but lack mature phenotype of adult primary human hepatocytes (PHH). Herein, we show that 3D culture of iPS-derived hepatocytes and their co-culture with human sinusoidal endothelial cells (sECs) to improve their maturity.

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

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.

Stable engineered vascular networks from human induced pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels.

Here, we describe an in vitro strategy to model vascular morphogenesis where human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) are encapsulated in peptide-functionalized poly(ethylene glycol) (PEG) hydrogels, either on standard well plates or within a passive pumping polydimethylsiloxane (PDMS) tri-channel microfluidic device. PEG hydrogels permissive towards cellular remodeling were fabricated using thiol-ene photopolymerization to incorporate matrix metalloproteinase (MMP)-degradable crosslinks and CRGDS cell adhesion peptide. Time lapse microscopy, immunofluorescence imaging, and RNA sequencing (RNA-Seq) demonstrated that iPSC-ECs formed vascular networks through mechanisms that were consistent with in vivo vasculogenesis and angiogenesis when cultured in PEG hydrogels. Migrating iPSC-ECs condensed into clusters, elongated into tubules, and formed polygonal networks through sprouting. Genes upregulated for iPSC-ECs cultured in PEG hydrogels relative to control cells on tissue culture polystyrene (TCP) surfaces included adhesion, matrix remodeling, and Notch signaling pathway genes relevant to in vivo vascular development. Vascular networks with lumens were stable for at least 14days when iPSC-ECs were encapsulated in PEG hydrogels that were polymerized within the central channel of the microfluidic device. Therefore, iPSC-ECs cultured in peptide-functionalized PEG hydrogels offer a defined platform for investigating vascular morphogenesis in vitro using both standard and microfluidic formats. STATEMENT OF SIGNIFICANCE: Human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) cultured in synthetic hydrogels self-assemble into capillary networks through mechanisms consistent with in vivo vascular morphogenesis.