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Extracellular matrix cues regulate the differentiation of pluripotent stem cell-derived endothelial cells.

ABSTRACT: The generation of endothelial cells (ECs) from human pluripotent stem cells (PSCs) has been a promising approach for treating cardiovascular diseases for several years. Human PSCs, particularly induced pluripotent stem cells (iPSCs), are an attractive source of ECs for cell therapy. Although there is a diversity of methods for endothelial cell differentiation using biochemical factors, such as small molecules and cytokines, the efficiency of EC production varies depending on the type and dose of biochemical factors. Moreover, the protocols in which most EC differentiation studies have been performed were in very unphysiological conditions that do not reflect the microenvironment of native tissue. The microenvironment surrounding stem cells exerts variable biochemical and biomechanical stimuli that can affect stem cell differentiation and behavior. The stiffness and components of the extracellular microenvironment are critical inducers of stem cell behavior and fate specification by sensing the extracellular matrix (ECM) cues, adjusting the cytoskeleton tension, and delivering external signals to the nucleus. Differentiation of stem cells into ECs using a cocktail of biochemical factors has been performed for decades. However, the effects of mechanical stimuli on endothelial cell differentiation remain poorly understood. This review provides an overview of the methods used to differentiate ECs from stem cells by chemical and mechanical stimuli. We also propose the possibility of a novel EC differentiation strategy using a synthetic and natural extracellular matrix.

SUBMITTER: Noh KM

PROVIDER: S-EPMC10330705 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Extracellular matrix cues regulate the differentiation of pluripotent stem cell-derived endothelial cells.

Noh Kyung Mu KM Park Soon-Jung SJ Moon Sung-Hwan SH Jung Seok Yun SY

Frontiers in cardiovascular medicine 20230626

The generation of endothelial cells (ECs) from human pluripotent stem cells (PSCs) has been a promising approach for treating cardiovascular diseases for several years. Human PSCs, particularly induced pluripotent stem cells (iPSCs), are an attractive source of ECs for cell therapy. Although there is a diversity of methods for endothelial cell differentiation using biochemical factors, such as small molecules and cytokines, the efficiency of EC production varies depending on the type and dose of ...[more]

PMID: 37435057

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Project description:UnlabelledRecent 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.Statement of significanceHuman 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:Gene delivery within hydrogel matrices can potentially direct mesenchymal stem cells (MSCs) towards a chondrogenic fate to promote regeneration of cartilage. Here, we investigated whether the mechanical properties of the hydrogel containing the gene delivery systems could enhance transfection and chondrogenic programming of primary human bone marrow-derived MSCs. We developed collagen-I-alginate interpenetrating polymer network hydrogels with tunable stiffness and adhesion properties. The hydrogels were activated with nanocomplexed SOX9 polynucleotides to direct chondrogenic differentiation of MSCs. MSCs transfected within the hydrogels showed higher expression of chondrogenic markers compared to MSCs transfected in 2D prior to encapsulation. The nanocomplex uptake and resulting expression of transfected SOX9 were jointly enhanced by increased stiffness and cell-adhesion ligand density in the hydrogels. Further, transfection of SOX9 effectively induced MSCs chondrogenesis and reduced markers of hypertrophy compared to control matrices. These findings highlight the importance of matrix stiffness and adhesion as design parameters in gene-activated matrices for regenerative medicine. STATEMENT OF SIGNIFICANCE: Gene-activated matrices (GAMs) are biodegradable polymer networks integrating gene therapies, and they are promising technologies for supporting tissue regeneration. Despite this interest, there is still limited information on how to rationally design these systems. Here, we provide a systematic study of the effect of matrix stiffness and cell adhesion ligands on gene transfer efficiency. We show that high stiffness and the presence of cell-binding sites promote transfection efficiency and that this result is related to more efficient internalization and trafficking of the gene therapies. GAMs with optimized mechanical properties can induce cartilage formation and result in tissues with better characteristics for articular cartilage tissue engineering as compared to previously described standard methods.

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.

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Extracellular matrix cues regulate the differentiation of pluripotent stem cell-derived endothelial cells.

Publications

Extracellular matrix cues regulate the differentiation of pluripotent stem cell-derived endothelial cells.

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