Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Shah, S. P.; Lee, Hyun‐Sook; Park, Young Hwan; Jeon, Eunkyeong; Chung, Hye Kyung; Lee, Eun Sang; Shim, Jae Hoon; Kang, Kyu‐Tae

doi:10.3791/60032

Cited by 10 publications

(13 citation statements)

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“…In order to investigate the function of T-DPSCs in endothelial sprouting, the 3D spheroid sprouting assay was performed as described previously [ 25 ]. Briefly, HUVECs and DPSCs or T-DPSCs were mixed 1:1 at a density of 4×10 4 cells/mL in α-MEM containing 20% Methocel (Sigma).…”

Section: Methodsmentioning

confidence: 99%

“…In this study, a well-established in vitro 3D co-culture collagen gel model was employed to mimic multiple stages of angiogenesis [ 25 ]. We aimed to investigate (1) the dynamic expression of growth factors in the process of DPSC differentiation into functional pericyte-like cells, (2) whether T-DPSCs function like pericytes stabilizing the newly formed blood vessels, and (3) whether VEGF-Ang-Tie2 signaling is involved in the crosstalk between pericyte-like DPSCs and ECs.…”

Section: Introductionmentioning

confidence: 99%

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DPSCs treated by TGF-β1 regulate angiogenic sprouting of three-dimensionally co-cultured HUVECs and DPSCs through VEGF-Ang-Tie2 signaling

et al. 2021

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Background Maintaining the stability and maturation of blood vessels is of paramount importance for the vessels to carry out their physiological function. Smooth muscle cells (SMCs), pericytes, and mesenchymal stem cells (MSCs) are involved in the maturation process of the newly formed vessels. The aim of this study was to investigate whether transforming growth factor beta 1 (TGF-β1) treatment could enhance pericyte-like properties of dental pulp stem cells (DPSCs) and how TGF-β1-treated DPSCs for 7 days (T-DPSCs) stabilize the newly formed blood vessels. Methods We utilized TGF-β1 to treat DPSCs for 1, 3, 5, and 7 days. Western blotting and immunofluorescence were used to analyze the expression of SMC markers. Functional contraction assay was conducted to assess the contractility of T-DPSCs. The effects of T-DPSC-conditioned media (T-DPSC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation and migration were examined by MTT, wound healing, and trans-well migration assay. Most importantly, in vitro 3D co-culture spheroidal sprouting assay was used to investigate the regulating role of vascular endothelial growth factor (VEGF)-angiopoietin (Ang)-Tie2 signaling on angiogenic sprouting in 3D co-cultured spheroids of HUVECs and T-DPSCs. Angiopoietin 2 (Ang2) and VEGF were used to treat the co-cultured spheroids to explore their roles in angiogenic sprouting. Inhibitors for Tie2 and VEGFR2 were used to block Ang1/Tie2 and VFGF/VEGFR2 signaling. Results Western blotting and immunofluorescence showed that the expression of SMC-specific markers (α-SMA and SM22α) were significantly increased after treatment with TGF-β1. Contractility of T-DPSCs was greater compared with that of DPSCs. T-DPSC-CM inhibited HUVEC migration. In vitro sprouting assay demonstrated that T-DPSCs enclosed HUVECs, resembling pericyte-like cells. Compared to co-culture with DPSCs, a smaller number of HUVEC sprouting was observed when co-cultured with T-DPSCs. VEGF and Ang2 co-stimulation significantly enhanced sprouting in HUVEC and T-DPSC co-culture spheroids, whereas VEGF or Ang2 alone exerted insignificant effects on HUVEC sprouting. Blocking Tie2 signaling reversed the sprouting inhibition by T-DPSCs, while blocking VEGF receptor (VEGFR) signaling boosted the sprouting inhibition by T-DPSCs. Conclusions This study revealed that TGF-β1 can induce DPSC differentiation into functional pericyte-like cells. T-DPSCs maintain vessel stability through Ang1/Tie2 and VEGF/VEGFR2 signaling.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

DPSCs treated by TGF-β1 regulate angiogenic sprouting of three-dimensionally co-cultured HUVECs and DPSCs through VEGF-Ang-Tie2 signaling

et al. 2021

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“…Studies using MSC-EC co-cultures have revealed, for example, that ECs regulate the MSCs into their osteogenic differentiation and maintaining their quiescence in a 3D cellular context [ 143 ]. Studies focusing on the effects of MSCs on ECs and angiogenesis, in turn, have relied both on spheroid co-cultures [ 144 , 145 ] and on spheroids constructed only from MSCs [ 146 , 147 , 148 ]. These studies were able to improve the therapeutic effect of MSCs on ischemia areas.…”

Section: Novel In Vitro Technologies and Their Potential For Applimentioning

confidence: 99%

“…A co-culture model of Shah el al. not only increased those factors but also generated sprout structures that might later be relevant to further angiogenic models [ 144 ].…”

Section: Novel In Vitro Technologies and Their Potential For Applimentioning

confidence: 99%

Mesenchymal Stem Cells as a Promising Cell Source for Integration in Novel In Vitro Models

et al. 2020

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The human-relevance of an in vitro model is dependent on two main factors—(i) an appropriate human cell source and (ii) a modeling platform that recapitulates human in vivo conditions. Recent years have brought substantial advancements in both these aspects. In particular, mesenchymal stem cells (MSCs) have emerged as a promising cell source, as these cells can differentiate into multiple cell types, yet do not raise the ethical and practical concerns associated with other types of stem cells. In turn, advanced bioengineered in vitro models such as microfluidics, Organs-on-a-Chip, scaffolds, bioprinting and organoids are bringing researchers ever closer to mimicking complex in vivo environments, thereby overcoming some of the limitations of traditional 2D cell cultures. This review covers each of these advancements separately and discusses how the integration of MSCs into novel in vitro platforms may contribute enormously to clinical and fundamental research.

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“…On the other hand, the extracellular matrix (ECM) and/or basement membrane and circulating blood may also play a major role. Various efforts at coculturing endothelial cells with different cell types are performed, but there is no in vitro assay currently available that covers all the components of this complex process [ 55 ].…”

Section: Angiogenesis Assaysmentioning

confidence: 99%

An Overview of In Vitro, In Vivo, and Computational Techniques for Cancer-Associated Angiogenesis Studies

Rahman

Tan

Othman

et al. 2020

BioMed Research International

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Angiogenesis is a crucial area in scientific research because it involves many important physiological and pathological processes. Indeed, angiogenesis is critical for normal physiological processes, including wound healing and embryonic development, as well as being a component of many disorders, such as rheumatoid arthritis, obesity, and diabetic retinopathies. Investigations of angiogenic mechanisms require assays that can activate the critical steps of angiogenesis as well as provide a tool for assessing the efficacy of therapeutic agents. Thus, angiogenesis assays are key tools for studying the mechanisms of angiogenesis and identifying the potential therapeutic strategies to modulate neovascularization. However, the regulation of angiogenesis is highly complex and not fully understood. Difficulties in assessing the regulators of angiogenic response have necessitated the development of an alternative approach. In this paper, we review the standard models for the study of tumor angiogenesis on the macroscopic scale that include in vitro, in vivo, and computational models. We also highlight the differences in several modeling approaches and describe key advances in understanding the computational models that contributed to the knowledge base of the field.

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Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Cited by 10 publications

References 0 publications

DPSCs treated by TGF-β1 regulate angiogenic sprouting of three-dimensionally co-cultured HUVECs and DPSCs through VEGF-Ang-Tie2 signaling

DPSCs treated by TGF-β1 regulate angiogenic sprouting of three-dimensionally co-cultured HUVECs and DPSCs through VEGF-Ang-Tie2 signaling

Mesenchymal Stem Cells as a Promising Cell Source for Integration in Novel In Vitro Models

An Overview of In Vitro, In Vivo, and Computational Techniques for Cancer-Associated Angiogenesis Studies

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