Dataset Information

Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells.

ABSTRACT: Endothelial cells derived from human pluripotent stem cells are a promising cell type for enhancing angiogenesis in ischemic cardiovascular tissues. However, our understanding of microenvironmental factors that modulate the process of endothelial differentiation is limited. We examined the role of combinatorial extracellular matrix (ECM) proteins on endothelial differentiation systematically using an arrayed microscale platform. Human pluripotent stem cells were differentiated on the arrayed ECM microenvironments for 5 days. Combinatorial ECMs composed of collagen IV?+?heparan sulfate?+?laminin (CHL) or collagen IV?+?gelatin?+?heparan sulfate (CGH) demonstrated significantly higher expression of CD31, compared to single-factor ECMs. These results were corroborated by fluorescence activated cell sorting showing a 48% yield of CD31⁺/VE-cadherin⁺ cells on CHL, compared to 27% on matrigel. To elucidate the signaling mechanism, a gene expression time course revealed that VE-cadherin and FLK1 were upregulated in a dynamically similar manner as integrin subunit ?3 (>50 fold). To demonstrate the functional importance of integrin ?3 in promoting endothelial differentiation, the addition of neutralization antibody inhibited endothelial differentiation on CHL-modified dishes by >50%. These data suggest that optimal combinatorial ECMs enhance endothelial differentiation, compared to many single-factor ECMs, in part through an integrin ?3-mediated pathway.

SUBMITTER: Hou L

PROVIDER: S-EPMC5529516 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells.

Hou Luqia L Kim Joseph J JJ Wanjare Maureen M Patlolla Bhagat B Coller John J Natu Vanita V Hastie Trevor J TJ Huang Ngan F NF

Scientific reports 20170726 1

Endothelial cells derived from human pluripotent stem cells are a promising cell type for enhancing angiogenesis in ischemic cardiovascular tissues. However, our understanding of microenvironmental factors that modulate the process of endothelial differentiation is limited. We examined the role of combinatorial extracellular matrix (ECM) proteins on endothelial differentiation systematically using an arrayed microscale platform. Human pluripotent stem cells were differentiated on the arrayed ECM ...[more]

PMID: 28747756

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Project description: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.

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:Integrins provide the primary link between mesenchymal stem cells (MSCs) and their surrounding extracellular matrix (ECM), with different integrin pairs having specificity for different ECM molecules or peptide sequences contained within them. It is widely acknowledged that the type of ECM present can influence MSC differentiation; however, it is yet to be determined how specific integrin-ECM interactions may alter this or how they change during differentiation. We determined that human bone marrow-derived mesenchymal stem cells (hMSCs) express a broad range of integrins in their undifferentiated state and show a dramatic, but transient, increase in the level of ?5 integrin on day 7 of osteogenesis and an increase in ?6 integrin expression throughout adipogenesis. We used a nonfouling polystyrene-block-poly(ethylene oxide)-copolymer (PS-PEO) surface to present short peptides with defined integrin-binding capabilities (RGD, IKVAV, YIGSR, and RETTAWA) to hMSCs and investigate the effects of such specific integrin-ECM contacts on differentiation. hMSCs cultured on these peptides displayed different morphologies and had varying abilities to differentiate along the osteogenic and adipogenic lineages. The peptide sequences most conducive to differentiation (IKVAV for osteogenesis and RETTAWA and IKVAV for adipogenesis) were not necessarily those that were bound by those integrin subunits seen to increase during differentiation. Additionally, we also determined that presentation of RGD, which is bound by multiple integrins, was required to support long-term viability of hMSCs. Overall we confirm that integrin-ECM contacts change throughout hMSC differentiation and show that surfaces presenting defined peptide sequences can be used to target specific integrins and ultimately influence hMSC differentiation. This platform also provides information for the development of biomaterials capable of directing hMSC differentiation for use in tissue engineering therapies.

Dataset Information

Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells.

Publications

Combinatorial Extracellular Matrix Microenvironments for Probing Endothelial Differentiation of Human Pluripotent Stem Cells.

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