BPS2025 - Changes in collective behavior, organization, and state of human induced pluripotent stem cell-derived endothelial cells exposed to shear stress revealed by 3D microscopy and quantitative image analysis

Restoring anatomical complexity of a left ventricle wall as a step toward bioengineering a human heart with human induced pluripotent stem cell-derived cardiac cells

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

Chronic nicotine impairs the angiogenic capacity of human induced pluripotent stem cell-derived endothelial cells in a murine model of peripheral arterial disease

An end-to-end deep convolutional neural network-based data-driven fusion framework for identification of human induced pluripotent stem cell-derived endothelial cells in photomicrographs

BackgroundElectronic-cigarette (e-cig) usage, particularly in the youth population, is a growing concern. It is known that e-cig causes endothelial dysfunction, which is a risk factor for the development of cardiovascular diseases; however, the mechanisms involved remain unclear. We hypothesized that long noncoding RNAs (lncRNAs) may play a role in e-cig-induced endothelial dysfunction.MethodsHere, we identified lncRNAs that are dysregulated in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following 24 h of e-cig aerosol extract treatment via microarray analysis. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analyses of the dysregulated mRNAs following e-cig exposure and constructed co-expression networks of the top 5 upregulated lncRNAs and the top 5 downregulated lncRNAs and the mRNAs that are correlated with them. Furthermore, the functional effects of knocking down lncRNA lung cancer-associated transcript 1 (LUCAT1) on EC phenotypes were determined as it was one of the significantly upregulated lncRNAs following e-cig exposure based on our profiling.Results183 lncRNAs and 132 mRNAs were found to be upregulated, whereas 297 lncRNAs and 413 mRNAs were found to be downregulated after e-cig exposure. We also observed that e-cig caused dysregulation of endothelial metabolism resulting in increased FAO activity, higher mitochondrial membrane potential, and decreased glucose uptake and glycolysis. These results suggest that e-cig alters EC metabolism by increasing FAO to compensate for energy deficiency in ECs. Finally, the knockdown of LUCAT1 prevented e-cig-induced EC dysfunction by maintaining vascular barrier, reducing reactive oxygen species level, and increasing migration capacity.ConclusionThis study identifies an expression profile of differentially expressed lncRNAs and several potential regulators and pathways in ECs exposed to e-cig, which provide insights into the regulation of lncRNAs and mRNAs and the role of lncRNA and mRNA networks in ECs associated e-cig exposure.

Introduction: Electronic-cigarette (e-cig) usage, particularly in the youth population, is a growing concern. It is known that e-cig causes endothelial dysfunction, which is a risk factor for the development of cardiovascular diseases; however, the mechanisms involved remain unclear. We hypothesized that long non-coding RNAs (lncRNAs), a novel class of non-coding RNA that has been shown to affect endothelial function, may play a role in e-cig-induced endothelial dysfunction. Methods and Results: We profiled lncRNAs that are dysregulated in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from healthy individuals following 24 hours of e-cig aerosol extract treatment. Using microarray analysis, we identified that 183 lncRNAs and 132 mRNAs were upregulated while 297 lncRNAs and 413 mRNAs were downregulated. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analyses of the mRNAs dysregulated by e-cig exposure followed by the functional characterization of the top 5 differentially expressed (DE) lncRNAs by co-expressed protein-coding genes analysis. Interestingly, the lncRNA-mRNA association analysis revealed several mRNAs associated with metabolic disorders. Subsequently, we found that e-cig caused dysregulation of endothelial metabolism resulting in increased fatty acid oxidation activity, higher mitochondrial membrane potential, and decreased glucose uptake and glycolysis. These results suggest that e-cig-induced altered EC metabolism and increased fatty acid oxidation compensate for energy deficiency in ECs. Conclusion: Collectively, the identification of potential regulators and pathways of the interaction between lncRNAs and mRNAs in ECs treated with e-cig provides novel insights into endothelial biology and could have a significant impact on our understanding of vascular complications upon e-cig exposure.

Microphysiological systems (MPS), or "organ-on-a-chip" platforms, aim to recapitulate in vivo physiology using small-scale in vitro tissue models of human physiology. While significant efforts have been made to create vascularized tissues, most reports utilize primary endothelial cells that hinder reproducibility. In this study, we report the use of human induced pluripotent stem cell-derived endothelial cells (iPS-ECs) in developing three-dimensional (3D) microvascular networks. We established a CDH5-mCherry reporter iPS cell line, which expresses the vascular endothelial (VE)-cadherin fused to mCherry. The iPS-ECs demonstrate physiological functions characteristic of primary endothelial cells in a series of in vitro assays, including permeability, response to shear stress, and the expression of endothelial markers (CD31, von Willibrand factor, and endothelial nitric oxide synthase). The iPS-ECs form stable, perfusable microvessels over the course of 14 days when cultured within 3D microfluidic devices. We also demonstrate that inhibition of TGF-β signaling improves vascular network formation by the iPS-ECs. We conclude that iPS-ECs can be a source of endothelial cells in MPS providing opportunities for human disease modeling and improving the reproducibility of 3D vascular networks.

Background: Peripheral artery disease (PAD) affects approximately 230 million people globally and chronic limb-threatening ischemia (CLTI) can lead to limb amputation. Human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) offer a promising source for PAD treatment. However, to date, regulatory criteria for the clinical application of hiPSC-ECs have not been established yet, and there have been no reports on preclinical studies involving hiPSC-ECs. This study aims to address this gap by investigating the feasibility, safety, and efficacy of clinical-grade hiPSC-ECs through a preclinical proof-of-concept analysis. Methods and Results: Clinical-grade hiPSC lines were established from the blood of three PAD patients using episomal plasmids. Their pluripotency was confirmed by assessing their expression of pluripotency markers through qRT-PCR and immunostaining, as well as their pluripotency in a teratoma assay. Furthermore, PAD-hiPSCs exhibited normal karyotypes. Subsequently, we differentiated PAD-hiPSCs into ECs, all of which displayed a cobble-stone EC morphology and expressed EC markers as confirmed by qRT-PCR and immunostaining. PAD-hiPSC-derived ECs also expressed CDH5 at a minimum of 98.4 ± 0.2% and VWF at 94.4 ± 1.3% by flow cytometry. PAD-hiPSC-ECs maintained their normal karyotypes and copy number variation across the genome, as determined by CGH array. Additionally, they exhibited endothelial characteristics such as tube formation in Matrigel and intracellular nitric oxide production. Administration of PAD-hiPSC-ECs into ischemic hindlimbs of both female and male mice led to improved blood flow recovery (~3.3 fold), reduced risk of limb loss (~8.8 ± 0.6%), and increased vascular density (~2.7 fold) compared to control groups. These engrafted hiPSC-ECs exhibited vessel-forming capacities, thereby contributing to neovascularization. More importantly, we evaluated the toxicity, biodistribution, and tumorigenic potential of these cells in immunodeficient nude mice over one year. The results demonstrated non-detection of tumorigenic cells in twelve organs and adverse events. With all the data, the use of hiPSC-ECs was approved for a clinical trial to treat PAD. Conclusions: Our preclinical proof-of-concept findings demonstrate, for the first time, the clinical compatibility of hiPSC-ECs derived from patients for autologous cell therapy for PAD. Our study further suggests regulatory guidance for the clinical development of hiPSC-ECs.

A key feature of peripheral arterial disease (PAD) is damage to endothelial cells (ECs), resulting in lower limb pain and restricted blood flow. Recent preclinical studies demonstrate that the transplantation of ECs via direct injection into the affected limb can result in significantly improved blood circulation. Unfortunately, the clinical application of this therapy has been limited by low cell viability and poor cell function. To address these limitations we have developed an injectable, recombinant hydrogel, termed SHIELD (Shear-thinning Hydrogel for Injectable Encapsulation and Long-term Delivery) for cell transplantation. SHIELD provides mechanical protection from cell membrane damage during syringe flow. Additionally, secondary in situ crosslinking provides a reinforcing network to improve cell retention, thereby augmenting the therapeutic benefit of cell therapy. In this study, we demonstrate the improved acute viability of human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following syringe injection delivery in SHIELD, compared to saline. Using a murine hind limb ischemia model of PAD, we demonstrate enhanced iPSC-EC retention in vivo and improved neovascularization of the ischemic limb based on arteriogenesis following transplantation of iPSC-ECs delivered in SHIELD.

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

Background: This study aimed to characterize the N6-methyladenosine epitranscriptomic profile induced by mono(2-ethylhexyl) phthalate (MEHP) exposure using a human-induced pluripotent stem cell-derived endothelial cell model. Methods: A multiomic approach was employed by performing RNA sequencing in parallel with an N6-methyladenosine-specific microarray to identify mRNAs, lncRNAs, and miRNAs affected by MEHP exposure. Results: An integrative multiomic analysis identified relevant biological features affected by MEHP, while functional assays provided a phenotypic characterization of these effects. Transcripts regulated by the epitranscriptome were validated with quantitative PCR and methylated RNA immunoprecipitation. Conclusion: The authors' profiling of the epitranscriptome expands the scope of toxicological insights into known environmental toxins to under surveyed cellular contexts and emerging domains of regulation and is, therefore, a valuable resource to human health.

Intratracheally injected human-induced pluripotent stem cell-derived pneumocytes and endothelial cells engraft in the distal lung and ameliorate emphysema in a rat model

Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) can contribute to elucidating the pathogenesis of heart and vascular diseases and developing their treatments. Their precise characteristics in fluid flow however remain unclear. Therefore, the aim of the present study is to characterise these features. We cultured three types of ECs in a microfluidic culture system: commercially available human iPS-ECs, human umbilical vein endothelial cells (HUVECs) and human umbilical artery endothelial cells (HUAECs). We then examined the mRNA expression levels of endothelial marker gene cluster of differentiation 31 (CD31), fit-related receptor tyrosine kinase (Flk-1), and the smooth muscle marker gene smooth muscle alpha-actin, and investigated changes in plasminogen activator inhibitor-1 (PAI-1) secretion and intracellular F-actin arrangement following heat stress. We also compared expressions of the arterial and venous marker genes ephrinB2 and EphB4, and the endothelial gap junction genes connexin (Cx) 37, 40, and 43 under fluidic shear stress to determine their arterial or venous characteristics. We found that iPS-ECs had similar endothelial marker gene expressions and exhibited similar increases in PAI-1 secretion under heat stress as HUVECs and HUAECs. In addition, F-actin arrangement in iPSC-ECs also responded to heat stress, as previously reported. However, they had different expression patterns of arterial and venous marker genes and Cx genes under different fluidic shear stress levels, showing that iPSC-ECs exhibit different characteristics from arterial and venous ECs. This microfluidic culture system equipped with variable shear stress control will provide an easy-to-use assay tool to examine characteristics of iPS-ECs generated by different protocols in various laboratories and contribute to basic and applied biomedical researches on iPS-ECs.

Phospholipid nanoparticles have been actively explored for biological and biomedical applications. These nanoparticles display excellent cargo encapsulation efficiency, high water dispersibility, and outstanding biocompatibility, rendering them highly attractive for cancer nanomedicine applications. While the promising utilizations of phospholipid nanoparticles for cancer theranostic have been widely documented, the cellular biophysical responses elicited by phospholipid nanoparticles have been less investigated. Notably, the effects of phospholipid nanoparticles on collective cancer cell behaviors have not been well delineated. In fact, most of the studies examining nanoparticle-cancer interactions have focused largely on the nanoparticle toxicity on individual cells, but paid little attention to other nanoparticle effects on multicellular systems and their collective behaviors. A better appreciation of the effects of nanoparticles on multicellular system dynamics is necessary as collective cancer cell behaviors are central to the regulation of various processes, particularly cancer invasion and metastasis. Motivated by this, we sought to interrogate the effects of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (DSPE-PEG) nanoparticles, which are some of the most commonly used phospholipid nanoparticles for cancer nanomedicine, on the collective behaviors of healthy and cancerous breast epithelial cell monolayers. DSPE-PEG nanoparticles with two sizes (i.e., 20 and 60 nm) and three surface functional groups (i.e., COOH, OCH3, and NH2) as well as three types of breast epithelial cell sheets with varying malignancy potential (i.e., MCF-10A, MCF-7, and MDA-MB-231) were used in this work. Utilizing a series of microscopy and molecular biology techniques and quantitative image analysis, we comprehensively examined the collective cell migratory dynamics in the absence and presence of DSPE-PEG nanoparticles. We noted that DSPE-PEG nanoparticles retarded the migration of the healthy MCF-10A epithelial cell sheets. In contrast, the cancerous MCF-7 and MDA-MB-231 cell sheets experienced accelerated collective migration in the presence of the nanoparticles. Moreover, these nanoparticles altered the migration directionality of the cancerous breast cell sheets. We ascribed the differential nanoparticle-modulated collective cell migratory behaviors to changes in the stiffness of the cell nuclei, cytoplasm, and cell-cell junctions, as well as the reorganization of the cellular actin filament networks after the nanoparticle-cell interactions. We anticipate that this study will provide a deeper insight into the nanomaterial-cancer interactions and aid the formulation of phospholipid nanoparticles for more effective and safer cancer theranostic and nanomedicine applications. Citation Format: Kenry, Bin Liu. Differential collective cell migratory behaviors modulated by phospholipid nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 266.

An integral enabler of the smart city vision is the ability to effectively model collective population behaviour. The realisation of sustainable smart mobility is underpinned by the effective modelling of the spatial movements of the population. Furthermore, it is also crucial to identify significant deviations in collective behaviour over time. For example, a change in urban mobility patterns would subsequently impact traffic management systems. This paper focuses on the issue of modelling the collective behaviour of a population by utilizing mobile phone data and investigates the ability to identify significant deviations in behaviour over time. Mobile phone data facilitates the inference of real social networks from their call data records (CDR). We use this data to model collective behaviour and apply change-point detection algorithms, a category of anomaly detection, in order to identify statistically significant changes in collective behaviour over time. The result off the empirical analysis demonstrate that modern change point detection can accurately identify change points with an R2 value of 0.9633.