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Combinatorial extracellular matrix microenvironments promote survival and phenotype of human induced pluripotent stem cell-derived endothelial cells in hypoxia.

ABSTRACT: Recent developments in cell therapy using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) hold great promise for treating ischemic cardiovascular tissues. However, poor post-transplantation viability largely limits the potential of stem cell therapy. Although the extracellular matrix (ECM) has become increasingly recognized as an important cell survival factor, conventional approaches primarily rely on single ECMs for in vivo co-delivery with cells, even though the endothelial basement membrane is comprised of a milieu of different ECMs. To address this limitation, we developed a combinatorial ECM microarray platform to simultaneously interrogate hundreds of micro-scale multi-component chemical compositions of ECMs on iPSC-EC response. After seeding iPSC-ECs onto ECM microarrays, we performed high-throughput analysis of the effects of combinatorial ECMs on iPSC-EC survival, endothelial phenotype, and nitric oxide production under conditions of hypoxia (1% O2) and reduced nutrients (1% fetal bovine serum), as is present in ischemic injury sites. Using automated image acquisition and analysis, we identified combinatorial ECMs such as collagen IV+gelatin+heparan sulfate+laminin and collagen IV+fibronectin+gelatin+heparan sulfate+laminin that significantly improved cell survival, nitric oxide production, and CD31 phenotypic expression, in comparison to single-component ECMs. These results were further validated in conventional cell culture platforms and within three-dimensional scaffolds. Furthermore, this approach revealed complex ECM interactions and non-intuitive cell behavior that otherwise could not be easily determined using conventional cell culture platforms. Together these data suggested that iPSC-EC delivery within optimal combinatorial ECMs may improve their survival and function under the condition of hypoxia with reduced nutrients.Human endothelial cells (ECs) derived from induced pluripotent stem cells (iPSC-ECs) are promising for treating diseases associated with reduced nutrient and oxygen supply like heart failure. However, diminished iPSC-EC survival after implantation into diseased environments limits their therapeutic potential. Since native ECs interact with numerous extracellular matrix (ECM) proteins for functional maintenance, we hypothesized that combinatorial ECMs may improve cell survival and function under conditions of reduced oxygen and nutrients. We developed a high-throughput system for simultaneous screening of iPSC-ECs cultured on multi-component ECM combinations under the condition of hypoxia and reduced serum. Using automated image acquisition and analytical algorithms, we identified combinatorial ECMs that significantly improved cell survival and function, in comparison to single ECMs. Furthermore, this approach revealed complex ECM interactions and non-intuitive cell behavior that otherwise could not be easily determined.

SUBMITTER: Hou L

PROVIDER: S-EPMC5045796 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Combinatorial extracellular matrix microenvironments promote survival and phenotype of human induced pluripotent stem cell-derived endothelial cells in hypoxia.

Hou Luqia L Coller John J Natu Vanita V Hastie Trevor J TJ Huang Ngan F NF

Acta biomaterialia 20160804

<h4>Unlabelled</h4>Recent developments in cell therapy using human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) hold great promise for treating ischemic cardiovascular tissues. However, poor post-transplantation viability largely limits the potential of stem cell therapy. Although the extracellular matrix (ECM) has become increasingly recognized as an important cell survival factor, conventional approaches primarily rely on single ECMs for in vivo co-delivery with cells, ev ...[more]

PMID: 27498178

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Project description:Unlabelled: Induced pluripotent stem cells (iPSCs) are new diagnostic and potentially therapeutic tools to model disease and assess the toxicity of pharmaceutical medications. A common limitation of cell lineages derived from iPSCs is a blunted phenotype compared with fully developed, endogenous cells. We examined the influence of novel three-dimensional bioartificial microenvironments on function and maturation of hepatocyte-like cells differentiated from iPSCs and grown within an acellular, liver-derived extracellular matrix (ECM) scaffold. In parallel, we also compared a bioplotted poly-l-lactic acid (PLLA) scaffold that allows for cell growth in three dimensions and formation of cell-cell contacts but is infused with type I collagen (PLLA-collagen scaffold) alone as a "deconstructed" control scaffold with narrowed biological diversity. iPSC-derived hepatocytes cultured within both scaffolds remained viable, became polarized, and formed bile canaliculi-like structures; however, cells grown within ECM scaffolds had significantly higher P450 (CYP2C9, CYP3A4, CYP1A2) mRNA levels and metabolic enzyme activity compared with iPSC hepatocytes grown in either bioplotted PLLA collagen or Matrigel sandwich control culture. Additionally, the rate of albumin synthesis approached the level of primary cryopreserved hepatocytes with lower transcription of fetal-specific genes, α-fetoprotein and CYP3A7, compared with either PLLA-collagen scaffolds or sandwich culture. These studies show that two acellular, three-dimensional culture systems increase the function of iPSC-derived hepatocytes. However, scaffolds derived from ECM alone induced further hepatocyte maturation compared with bioplotted PLLA-collagen scaffolds. This effect is likely mediated by the complex composition of ECM scaffolds in contrast to bioplotted scaffolds, suggesting their utility for in vitro hepatocyte assays or drug discovery.SignificanceThrough the use of novel technology to develop three-dimensional (3D) scaffolds, the present study demonstrated that hepatocyte-like cells derived via induced pluripotent stem cell (iPSC) technology mature on 3D extracellular matrix scaffolds as a result of 3D matrix structure and scaffold biology. The result is an improved hepatic phenotype with increased synthetic and catalytic potency, an improvement on the blunted phenotype of iPSC-derived hepatocytes, a critical limitation of iPSC technology. These findings provide insight into the influence of 3D microenvironments on the viability, proliferation, and function of iPSC hepatocytes to yield a more mature population of cells for cell toxicity studies and disease modeling.

Project description:Normal epithelium exists within a dynamic extracellular matrix (ECM) that is tuned to regulate tissue specific epithelial cell function. As such, ECM contributes to tissue homeostasis, differentiation, and disease, including cancer. Though it is now recognized that the functional unit of normal and transformed epithelium is the epithelial cell and its adjacent ECM, we lack a basic understanding of tissue-specific ECM composition and abundance, as well as how physiologic changes in ECM impact cancer risk and outcomes. While traditional proteomic techniques have advanced to robustly identify ECM proteins within tissues, methods to determine absolute abundance have lagged. Here, with a focus on tissues relevant to breast cancer, we utilize mass spectrometry methods optimized for absolute quantitative ECM analysis. Employing an extensive protein extraction and digestion method, combined with stable isotope labeled Quantitative conCATamer (QconCAT) peptides that serve as internal standards for absolute quantification of protein, we quantify 98 ECM, ECM-associated, and cellular proteins in a single analytical run. In rodent models, we applied this approach to the primary site of breast cancer, the normal mammary gland, as well as a common and particularly deadly site of breast cancer metastasis, the liver. We find that mammary gland and liver have distinct ECM abundance and relative composition. Further, we show mammary gland ECM abundance and relative compositions differ across the reproductive cycle, with the most dramatic changes occurring during the pro-tumorigenic window of weaning-induced involution. Combined, this work suggests ECM candidates for investigation of breast cancer progression and metastasis, particularly in postpartum breast cancers that are characterized by high metastatic rates. Finally, we suggest that with use of absolute quantitative ECM proteomics to characterize tissues of interest, it will be possible to reconstruct more relevant in vitro models to investigate tumor-ECM dynamics at higher resolution.

Dataset Information

Combinatorial extracellular matrix microenvironments promote survival and phenotype of human induced pluripotent stem cell-derived endothelial cells in hypoxia.

Publications

Combinatorial extracellular matrix microenvironments promote survival and phenotype of human induced pluripotent stem cell-derived endothelial cells in hypoxia.

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