Fabrication and Paracrine Effect Research of Bone Marrow-Derived Endothelial Progenitor Cell Sheet

Abstract Background: The release of a wide array of endothelial progenitor cell (EPC) sheet-secreted paracrine factors is central to the mechanism by which these cells contribute to tissue repair. The purpose of this study was to fabricate BM-EPC sheet and conduct preliminary investigation on the paracrine effect of SDF-1ɑ about the role of the stromal cell-derived factor-1α (SDF-1α)/CXCR4 axis in the form of tubular structures from BM-EPCs sheet.Methods: EPCs derived from rat bone marrow (BM-EPC) were identified and isolated by the cell-surface markers CD34/CD133/VE-cadherin/KDR using flow cytometry, as well as by dual affinity for acLDL and UEA-1. Single-cell suspensions were seeded on temperature-responsive cell culture dishes. After 7 days of incubation, the BM-EPC cells were easily harvested as cell sheets, and a series of biochemical experiments were performed in vitro. The expression levels of SDF-1α/CXCR4 axis-associated genes and proteins were examined using RT‑qPCR and western blot analysis, and enzyme-linked immunosorbent assay (ELISA) was applied to determine the concentrations of VEGF, EGF and SDF-1α in the cell culture medium.Results: The BM-EPC cell sheets were successfully harvested. Moreover, BM-EPC cell sheets have superior proliferation and tube formation activity when compare with single cell suspension. When capillary-like tube were formed from EPCs sheets, the releasing of paracrine factors such as VEGF, EGF, and SDF-1ɑ were increasing. To drive the tube formation of BM-EPCs sheets, our mechanism research showed that the activation of PI3K/AKT/eNOS pathway since the phosphorylation of protein CXCR, PI3K, AKT and eNOS were increasing. Conclusion: BM-EPC cell sheets have superior proliferation and tube formation activity that can be used for tissue repair. The strong ability to secrete paracrine factors was be related to the SDF-1α/CXCR4 axis through PI3K/AKT/eNOS pathway.

Download Full-text

Fabrication and Paracrine Effect Research of Bone Marrow-Derived Endothelial Progenitor Cell Sheet

10.21203/rs.3.rs-97639/v2 ◽

2020 ◽

Author(s):

Fenlong Xue ◽

Yuanfeng Xin ◽

Yunpeng Bai ◽

Yiyao Jiang ◽

Jianhsi Liu ◽

...

Keyword(s):

Cell Culture ◽

Bone Marrow ◽

Single Cell ◽

Endothelial Progenitor Cell ◽

Tissue Repair ◽

Progenitor Cell ◽

Tube Formation ◽

Cell Sheets ◽

Paracrine Factors ◽

Cxcr4 Axis

Abstract Background: The release of a wide array of endothelial progenitor cell (EPC) sheet-secreted paracrine factors is central to the mechanism by which these cells contribute to tissue repair. The purpose of this study was to fabricate BM-EPC sheet and conduct preliminary investigation on the paracrine effect of SDF-1ɑ about the role of the stromal cell-derived factor-1α (SDF-1α)/CXCR4 axis in the form of tubular structures from BM-EPCs sheet.Methods: EPCs derived from rat bone marrow (BM-EPC) were identified and isolated by the cell-surface markers CD34/CD133/VE-cadherin/KDR using flow cytometry, as well as by dual affinity for acLDL and UEA-1. Single-cell suspensions were seeded on temperature-responsive cell culture dishes. After 7 days of incubation, the BM-EPC cells were easily harvested as cell sheets, and a series of biochemical experiments were performed in vitro. The expression levels of SDF-1α/CXCR4 axis-associated genes and proteins were examined using RT‑qPCR and western blot analysis, and enzyme-linked immunosorbent assay (ELISA) was applied to determine the concentrations of VEGF, EGF and SDF-1α in the cell culture medium.Results: The BM-EPC cell sheets were successfully harvested. Moreover, BM-EPC cell sheets have superior proliferation and tube formation activity when compare with single cell suspension. When capillary-like tube were formed from EPCs sheets, the releasing of paracrine factors such as VEGF, EGF, and SDF-1ɑ were increasing. To drive the tube formation of BM-EPCs sheets, our mechanism research showed that the activation of PI3K/AKT/eNOS pathway since the phosphorylation of protein CXCR, PI3K, AKT and eNOS were increasing.Conclusion: BM-EPC cell sheets have superior proliferation and tube formation activity that can be used for tissue repair. The strong ability to secrete paracrine factors was be related to the SDF-1α/CXCR4 axis through PI3K/AKT/eNOS pathway.

Download Full-text

Construction and a preliminary study of paracrine effect of bone marrow-derived endothelial progenitor cell sheet

Cell and Tissue Banking ◽

10.1007/s10561-021-09932-w ◽

2021 ◽

Author(s):

Fenlong Xue ◽

Yunpeng Bai ◽

Yiyao Jiang ◽

Jianshi Liu ◽

Kaitao Jian

Keyword(s):

Bone Marrow ◽

Growth Factor ◽

Single Cell ◽

Endothelial Progenitor Cell ◽

Progenitor Cell ◽

Tube Formation ◽

Cell Sheets ◽

Endothelial Progenitor ◽

Paracrine Factors ◽

Cxcr4 Axis

AbstractThe release of paracrine factors from endothelial progenitor cell (EPC) sheet is a central mechanism of tissue repair. The purpose of this study was to constuct the rat bone marrow derived-endothelial progenitor cell (BM-EPCs) sheet and investigate invest the role of stromal cell-derived factor-1α (SDF-1α)/CXCR4 axis in the biological function of BM-EPCs sheet. BM-EPC cells were identified by the cell-surface markers-CD34/CD133/VE-cadherin/KDR using flow cytometry and dual affinity for acLDL and UEA-1. After 7 days of incubation, the BM-EPC single-cell suspensions were seeded on thermo-sensitive plate to harvest the BM-EPC cell sheets. The expression levels of SDF-1α/CXCR4 axis-associated genes and proteins were examined using RT-qPCR and western blot analysis, and enzyme-linked immunosorbent assay (ELISA) was applied to determine the concentration of vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and SDF-1α in the cell culture medium. The BM-EPC cell sheets were successfully harvested. Moreover, BM-EPC cell sheets have superior migration and tube formation activity when compared with single cell suspension. When capillary-like tube were formed from EPCs sheets, the releasing of paracrine factors such as VEGF, EGF and SDF-1α were increased. To reveal the mechanism of tube formation of BM-EPCs sheets, our research showed that the activation of PI3K/AKT/eNOS pathway was involved in the process, because the phosphorylation of CXCR, PI3K, AKT and eNOS were increased. BM-EPC cell sheets have superior paracrine and tube formation activity than the BM-EPC single-cell. The strong ability to secrete paracrine factors was be potentially related to the SDF-1α/CXCR4 axis through PI3K/AKT/eNOS pathway.

Download Full-text

Layered smooth muscle cell–endothelial progenitor cell sheets derived from the bone marrow augment postinfarction ventricular function

Journal of Thoracic and Cardiovascular Surgery ◽

10.1016/j.jtcvs.2017.04.081 ◽

2017 ◽

Vol 154 (3) ◽

pp. 955-963 ◽

Cited By ~ 8

Author(s):

Yasuhiro Shudo ◽

Andrew B. Goldstone ◽

Jeffrey E. Cohen ◽

Jay B. Patel ◽

Michael S. Hopkins ◽

...

Keyword(s):

Bone Marrow ◽

Smooth Muscle ◽

Smooth Muscle Cell ◽

Ventricular Function ◽

Muscle Cell ◽

Endothelial Progenitor Cell ◽

Progenitor Cell ◽

Cell Sheets ◽

Endothelial Progenitor

Download Full-text

Single-cell RNA sequencing of human fetal epicardium reveals novel markers and regulators of EMT

European Heart Journal ◽

10.1093/ehjci/ehaa946.3574 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

T.J Streef ◽

T Van Herwaarden ◽

A.M Smits ◽

M.J Goumans

Keyword(s):

Cell Culture ◽

Single Cell ◽

Rna Sequencing ◽

Public Institution ◽

Funding Source ◽

Injury Response ◽

Post Injury ◽

Paracrine Factors ◽

Adult Heart ◽

Single Cell Rna Sequencing

Abstract Background The heart is covered by the epicardium, consisting of epithelial cells and a mesenchymal layer. The epicardium has been shown to be essential during cardiac development by contributing cells through epithelial-to-mesenchymal transition (EMT) and the secretion of paracrine factors. In the adult, the epicardium conveys a cardioprotective response after myocardial infarction, albeit suboptimal compared to the epicardial contribution to heart development. Although the developing epicardium has been characterised in mice and zebrafish, knowledge on the human fetal epicardium derives mostly from cell culture models. Therefore, direct analysis of the human fetal epicardium is vital as it provides new insights into the cellular and biochemical interactions within the developing heart, which can potentially contribute to enhancing the post-injury response. Aim To study the human fetal epicardium using single-cell RNA sequencing (scRNA seq) in order to determine its cellular compositionThe data are further explored to e.g.identify regulators of epicardial EMT. Methods Epicardial layers were isolated from four fetal human hearts (14–15 weeks gestation, obtained under informed consent and according to local ethical approval). Tissue was digested, and single live cells were sorted into 384-wells plates and sequenced. Data analysis was performed using R-packages RaceID3 and StemID2. Findings were validated using qPCR and immunohistochemistry. Results Analysis of 2024 cells reveals a clear clustering of the epicardial epithelium and the mesenchymal population. Importantly, we found that “classical” markers, such as Wilms' Tumor 1 and T-box transcription factor 18, are not specific enough to reliably identify the epicardium, but our analysis has provided markers that do allow for robust identification of the epicardium. Additionally, we were able to identify epicardial subpopulations based on their expression profile, and we are currently investigating these using immunohistochemistry in human fetal and adult heart tissue sections. To establish the regulation of epicardial activation we are focussing on the process of EMT within our dataset using RaceID2. From our analysis, several regulators of epicardial EMT are proposed that will be followed up on in vitro. Conclusions We identify various novel markers of the fetal epithelial epicardium, as well as characterizing markers of the mesenchymal layer. We also identified novel factors involved in epicardial EMT, and these are currently being validated in our cell-culture model. These data can provide new insights into the post-injury response in the adult heart. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Dutch Heart Foundation

Download Full-text