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

Zhang, Jue; Schwartz, Michael P.; Hou, Zhonggang; Bai, Yongsheng; Ardalani, Hamisha; Swanson, Scott; Steill, John; Ruotti, Victor; Elwell, Angela L.; Nguyen, Bao Kim; Bolin, Jennifer M.; Stewart, Ron; Thomson, James A.; Murphy, William L.

doi:10.1016/j.stemcr.2017.02.014

Cited by 42 publications

(61 citation statements)

References 72 publications

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“…4 One comprehensive genome-wide study comparing iPSC-ECs grown in 2D or 3D substrates revealed that iPSC-ECs developed in 2D adopt a more proliferative phenotype characterized by increased focal adhesion kinase (FAK)–extracellular signal-regulated kinase (ERK) activity, in which inhibition of ERK signaling increased the expression of vasculature development genes. 57 Therefore, it is possible that the difference in signal transduction dynamics between integrins and cell adhesion kinases in 2D versus 3D contributes highly to endothelial differentiation capacity and cellular organization.…”

Section: Discussionmentioning

confidence: 99%

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Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells

et al. 2017

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INTRODUCTION Human 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. METHODS Human 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. RESULTS The 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. CONCLUSION Together, 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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Section: Discussionmentioning

confidence: 99%

“…33,57 We hypothesized that endothelial differentiation directly within topographically aligned 3D scaffolds may be a facile one-step approach to generate iPSC-ECs with aligned network-like organization to mimic that of myocardial microvasculature.…”

Section: Introductionmentioning

confidence: 99%

Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells

et al. 2017

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“…5f) and GSE93511(2D_MG_H1EC and 2D_MG_HUVEC ) ( Fig. 5e) [26], these genes including CD43(SPN), CD45 (PTPRC) and PROM1 which express in early-EPC have some expression. Thus, we think conservatively that normal EPCs, IPAH-EPCs and ESC/iPSC-ECs we obtained have characteristics of early EPCs.…”

Section: Results Showed Ecs On Day 7 With Higher Expression Level Of mentioning

confidence: 94%

“…A high-efficiency protocol for deriving ECs from ESCs or iPSCs has been published [26], we optimized the protocol according to other protocols [15,25,29], we seeded about 3 × 10 4 cells/well in 12-well plate using 10 µM Y27632 and 10 ng/mL BMP4 for the three days (Fig. 1a, b and c).…”

Section: A Modified Protocol For Generating Ecs From Human Escs or Ipscsmentioning

confidence: 99%

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Using human pluripotent stem cell differentiated endothelial cells to study the differential regulation in PAH

Qin¹,

Yang²

2020

Preprint

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Aims Endothelial cells (ECs) have been applied in clinic to treat pulmonary arterial hypertension (PAH), a disease characterized by disordered pulmonary vasculature. However, lack of enough transplantable cells before the deterioration of patient condition is current limitation to apply cell therapy in cardiovascular diseases. So, we thought it necessary to continue to differentiate embryo stem cells (ESCs)/induced-pluripotent stem cells (iPSCs) into endothelial cells (ECs) and identify their characteristics. Methods and results Comparing previous reported methods of human pluripotent cell differentiation toward vascular cells/ Hemogenic Endothelial cells/ endothelial colony-forming cells, we established a highly efficient differentiation protocol to get cells that match phenotype of isolated ECs from health donors. This protocol, including two stages, early mesoderm endothelial progenitor stage and EC marker expression stage. In the first stage, Rock inhibitor Y27632 and DMSO plays an important role in inducing-APLNR + mesoderm and promotes EC differentiation potential; later on SB431542 and BMP4 drives cells toward EC lineage. Meanwhile, an improved protocol with chemically-defined medium (CDM) has similar differentiation efficiency,again demonstrating that a large number of clinically needed cells could be obtained with simple factors. ESC/iPSC-ECs, normal EPCs and IPAH-EPCs have the characteristics of early EPCs marked by CD133 and mesenchymal stem cells. Microarray analysis further revealed IPAH-derived EPCs features of rapid proliferation and weak immune regulation. At last, a model Zebrafish xenograft was utilized to assess the functionality of differentiated ESC/iPSC-ECs. ConclusionsWe established a highly efficient differentiation protocol to get ESC/iPSC-ECs with characteristics of phenotype and molecular matched with early-EPCs from health donors, and revealed the molecular pathogenesis in PAH.

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Quantitative Label‐Free Imaging of 3D Vascular Networks Self‐Assembled in Synthetic Hydrogels

Kaushik

Gil

Torr

et al. 2018

Adv Healthcare Materials

Self Cite

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Vascularization is an important strategy to overcome diffusion limits and enable the formation of complex, physiologically relevant engineered tissues and organoids. Self-assembly is a technique to generate in vitro vascular networks, but engineering the necessary network morphology and function remains challenging. Here, autofluorescence multiphoton microscopy (aMPM), a label-free imaging technique, is used to quantitatively evaluate in vitro vascular network morphology. Vascular networks are generated using human embryonic stem cell–derived endothelial cells and primary human pericytes encapsulated in synthetic poly(ethylene glycol)-based hydrogels. Two custom-built bioreactors are used to generate distinct fluid flow patterns during vascular network formation: recirculating flow or continuous flow. aMPM is used to image these 3D vascular networks without the need for fixation, labels, or dyes. Image processing and analysis algorithms are developed to extract quantitative morphological parameters from these label-free images. It is observed with aMPM that both bioreactors promote formation of vascular networks with lower network anisotropy compared to static conditions, and the continuous flow bioreactor induces more branch points compared to static conditions. Importantly, these results agree with trends observed with immunocytochemistry. These studies demonstrate that aMPM allows label-free monitoring of vascular network morphology to streamline optimization of growth conditions and provide quality control of engineered tissues.

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

Cited by 42 publications

References 72 publications

Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells

Microfibrous Scaffolds Enhance Endothelial Differentiation and Organization of Induced Pluripotent Stem Cells

Using human pluripotent stem cell differentiated endothelial cells to study the differential regulation in PAH

Quantitative Label‐Free Imaging of 3D Vascular Networks Self‐Assembled in Synthetic Hydrogels

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