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Micropattern size-dependent endothelial differentiation from a human induced pluripotent stem cell line.

ABSTRACT: The multifaceted extracellular milieu presents biochemical and biophysical stimuli that influence stem cell differentiation. Two-dimensional (2D) micropatterned substrates allow the presentation of these cues in spatially defined geometries that have been demonstrated to guide stem cell fate decisions. Leveraging stem cells to reconstruct microvasculature, made up of an inner lining of endothelial cells (ECs) supported by pericytes, is critical to tissue-engineering advances; thus, methods to improve endothelial differentiation efficiency are vital to these efforts. In this study, we examine the hypothesis that the diameter of micropatterned islands influences endothelial differentiation from human induced pluripotent stem cells (hiPSCs). Comparing island diameters of 80, 140, 225 and 500 µm, we found that co-cultures of control ECs and pericytes did not yield variable ratios of cell types; however, when hiPSCs were differentiated toward a bicellular population of ECs and pericytes on these varying micropattern feature sizes, we found that smaller islands promoted EC differentiation efficiency, yielding a derived population composed of 70% ECs, which exhibited a greater sprouting propensity. Differentiation on the largest feature size exhibited a smaller EC yield, similar to that on non-patterned substrates. Taken together, these data demonstrate that micropatterned islands of varying diameters can be used to modulate EC differentiation efficiency. Copyright © 2015 John Wiley & Sons, Ltd.

SUBMITTER: Kusuma S

PROVIDER: S-EPMC4520778 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Micropattern size-dependent endothelial differentiation from a human induced pluripotent stem cell line.

Kusuma Sravanti S Smith Quinton Q Facklam Amanda A Gerecht Sharon S

Journal of tissue engineering and regenerative medicine 20150130 3

The multifaceted extracellular milieu presents biochemical and biophysical stimuli that influence stem cell differentiation. Two-dimensional (2D) micropatterned substrates allow the presentation of these cues in spatially defined geometries that have been demonstrated to guide stem cell fate decisions. Leveraging stem cells to reconstruct microvasculature, made up of an inner lining of endothelial cells (ECs) supported by pericytes, is critical to tissue-engineering advances; thus, methods to im ...[more]

PMID: 25641688

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Project description:BackgroundEndothelial cells (ECs) are essential regulators of the vasculature, lining arteries, veins, and capillary beds. While all ECs share a number of structural and molecular features, heterogeneity exists depending on their resident tissue. ECs lining the choriocapillaris in the human eye are lost early in the pathogenesis of age-related macular degeneration (AMD), a common and devastating form of vision loss. In order to study the mechanisms leading to choroidal endothelial cell (CEC) loss and to develop reagents for repairing the choroid, a reproducible in vitro model, which closely mimic CECs, is needed. While a number of protocols have been published to direct induced pluripotent stem cells (iPSCs) into ECs, the goal of this study was to develop methods to differentiate iPSCs into ECs resembling those found in the human choriocapillaris specifically.MethodsWe transduced human iPSCs with a CDH5p-GFP-ZEO lentiviral vector and selected for transduced iPSCs using blasticidin. We generated embryoid bodies (EBs) from expanded iPSC colonies and transitioned from mTESR™1 to EC media. One day post-EB formation, we induced mesoderm fate commitment via addition of BMP-4, activin A, and FGF-2. On day 5, EBs were adhered to Matrigel-coated plates in EC media containing vascular endothelial cell growth factor (VEGF) and connective tissue growth factor (CTGF) to promote CEC differentiation. On day 14, we selected for CECs using either zeocin resistance or anti-CD31 MACS beads. We expanded CECs post-selection and performed immunocytochemical analysis of CD31, carbonic anhydrase IV (CA4), and RGCC; tube formation assays; and transmission electron microscopy to access vascular function.ResultsWe report a detailed protocol whereby we direct iPSC differentiation toward mesoderm and utilize CTGF to specify CECs. The CDH5p-GFP-ZEO lentiviral vector facilitated the selection of iPSC-derived ECs that label with antibodies directed against CD31, CA4, and RGCC; form vascular tubes in vitro; and migrate into empty choroidal vessels. CECs selected using either antibiotic selection or CD31 MACS beads showed similar characteristics, thereby making this protocol easily reproducible with or without lentiviral vectors.ConclusionECs generated following this protocol exhibit functional and biochemical characteristics of CECs. This protocol will be useful for developing in vitro models toward understanding the mechanisms of CEC loss early in AMD.

Project description:IntroductionHuman induced pluripotent stem cells (iPSCs) are a promising source of endothelial cells (iPSC-ECs) for engineering three-dimensional (3D) vascularized cardiac tissues. To mimic cardiac microvasculature, in which capillaries are oriented in parallel, we hypothesized that endothelial differentiation of iPSCs within topographically aligned 3D scaffolds would be a facile one-step approach to generate iPSC-ECs as well as induce aligned vascular organization.MethodsHuman iPSCs underwent endothelial differentiation within electrospun 3D polycaprolactone (PCL) scaffolds having either randomly oriented or parallel-aligned microfibers. Using transcriptional, protein, and endothelial functional assays, endothelial differentiation was compared between conventional two-dimensional (2D) films and 3D scaffolds having either randomly oriented or aligned microfibers. Furthermore, the role of parallel-aligned microfiber patterning on the organization of vessel-like networks was assessed.ResultsThe cells in both the randomly oriented and aligned 3D scaffolds demonstrated an 11-fold upregulation in gene expression of the endothelial phenotypic marker, CD31, compared to cells on 2D films. This upregulation corresponded to >3-fold increase in CD31 protein expression in 3D scaffolds, compared to 2D films. Concomitantly, other endothelial phenotypic markers including CD144 and endothelial nitric oxide synthase also showed significant transcriptional upregulation in 3D scaffolds by >7-fold, compared to 2D films. Nitric oxide production, which is characteristic of endothelial function, was produced 4-fold more abundantly in 3D scaffolds, compared to on 2D PCL films. Within aligned scaffolds, the iPSC-ECs displayed parallel-aligned vascular-like networks with 70% longer branch length, compared to cells in randomly oriented scaffolds, suggesting that fiber topography modulates vascular network-like formation and patterning.ConclusionTogether, these results demonstrate that 3D scaffold structure promotes endothelial differentiation, compared to 2D substrates, and that aligned topographical patterning induces anisotropic vascular network organization.

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Micropattern size-dependent endothelial differentiation from a human induced pluripotent stem cell line.

Publications

Micropattern size-dependent endothelial differentiation from a human induced pluripotent stem cell line.

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