Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Immunomagnetic Isolation of the Vascular Wall-Resident CD34⁺ Stem Cells from Mice

Published: December 22, 2023

doi:

Chuting Han, Cheng Xie, Qingya Dang, Xiaolin Zhang, Chunshu Li, Yan Yang, Jun Cheng, Pengyun Li

¹Key Laboratory of Medical Electrophysiology of Ministry of Education and Medical Electrophysiological Key Laboratory of Sichuan Province, Institute of Cardiovascular Research,Southwest Medical University

Summary

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34⁺ stem cells (CD34⁺ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Abstract

Resident CD34⁺ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34⁺ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34⁺ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca²⁺ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 × 10⁷. Subsequently, CD34⁺ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34⁺ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca²⁺ release and external Ca²⁺ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34⁺ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Introduction

The vascular wall plays a pivotal role in vascular development, homeostatic regulation, and the progression of vascular diseases. In recent years, numerous studies have unveiled the presence of various stem cell lineages in arteries. In 2004, Professor Qingbo Xu's group first reported the existence of vascular stem/progenitor cells in the periphery of the adult vascular wall, expressing CD34, Sca-1, c-kit, and Flk-1¹. These vascular stem cells exhibit multidirectional differentiation and proliferation potential. Under normal conditions, they remain relatively quiescent; however, when activated by specific factors, they can differentiate into smooth muscle cells, endothelial cells, and fibroblasts. Alternatively, they can regulate the perivascular matrix and microvessel formation through paracrine effects to promote the repair or remodeling of injured vessels²^,³^,⁴^,⁵^,⁶. Recently, resident CD34⁺ stem cells in the vascular wall were found to play a role in endothelial cell regeneration after femoral artery guidewire injury². Consequently, the isolation and quantification of CD34⁺ VW-SCs and the examination of the basic biological characteristics of CD34⁺ stem cells are crucial for further studying the signal pathways involved in the regulation of CD34⁺ VW-SCs.

Various methods for cell separation are currently available, including techniques based on cell culture characteristics or physical properties of cells such as density gradient centrifugation, which results in sorted cells containing many non-target cells and relatively low purity⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹². Another commonly used technique is fluorescence/magnetic-assisted cell sorting. Fluorescence-activated cell sorting (FACS) is a complex system with high technical requirements, and it is relatively expensive, time-consuming, and potentially affects the activity of sorted cells¹³^,¹⁴. However, magnetic-activated cell sorting (MACS) is more efficient and convenient, with a high recovery rate and cell activity and less impact on downstream applications⁸. Therefore, in this protocol, we applied MACS to purify CD34⁺ VW-SCs and further identified the cells by flow cytometry. The establishment of MACS-based isolation methods for studying vascular wall stem cells would be invaluable. Firstly, it permits experimental genetic and cell biological studies. Secondly, efficient isolation and culture of vascular wall resident stem cells allow systematic assessment and screening of signaling factors regulating stem cell functions. Thirdly, identification of crucial phenotypes in stem cells provides important 'quality control' in assessing cell status. Thus, identifying methods to purify could be useful for similar applications to analogous stem cells derived from vessels.

This report provides a detailed demonstration of a stable and reliable method for the culture of CD34⁺ VW-SCs, including cell identification and functional assessment performed by flow cytometry, immunofluorescence staining, and Ca²⁺ signaling measurement. This study provides a basis for further in-depth research on the function of CD34⁺ VW-SCs and their regulatory mechanisms in physiological and pathological conditions.

Protocol

This study was approved, and the animals were handled in accordance with the Guidelines for the Management and Use of Laboratory Animals in China. The research strictly adhered to the ethical requirements of animal experiments, with approval from the Animal Ethics Committee (Approval Number: SWMU2020664). Eight-week-old healthy C57BL/6 mice of either gender, weighing between 18-20 g, were utilized for the present study. The animals were housed at the Laboratory Animal Center of Southwest Medical University (SWMU). <p…

Representative Results

Isolation and purification of CD34+ VW-SCs High purity of CD34+ VW-SCs is obtained from the adventitia of the mouse aortic and mesenteric artery by tissue attachment and magnetic microbead sorting. The percentage of CD34+ cells in the vessel wall is generally 10%-30%. Flow cytometry confirms that the purity of CD34+ cells obtained by magnetic bead sorting is more than 90% (Figure 1A). Cellular immunofluorescence staining show…

Discussion

This study provides a quick and convenient method for obtaining functional CD34⁺ VW-SCs from the aorta and mesenteric arteries of mice. CD34⁺ VW-SCs obtained by this method have proliferative activity and multidirectional differentiation properties. Triphosphate inositol 1,4,5-trisphosphate receptors (IP₃Rs), ryanodine receptors (RyRs), and store-operated calcium channels mediate Ca²⁺ release and entry in CD34⁺ VW-SCs. The establishment of this technique will lay the…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was funded by grants from National Natural Science Foundation of China (No. 82070502, 31972909, 32171099), the Sichuan Science and Technology Program of Sichuan Province (23NSFSC0576, 2022YFS0607). The authors would like to thank Qingbo Xu from Zhejiang University for help with the cell culture, and the authors acknowledge the scientific and technical assistance of the flow cytometry platform in Southwest Medical University.

Materials

2% gelatin solution	Sigma	G1393
Anti-CD31 antibody	R&D	AF3628
Anti-CD34 antibody	Abcam	ab81289
Anti-c-kit antibody	CST	77522
Anti-FITC MicroBeads	Miltenyi Biotec	130-048-701
Anti-FITC MicroBeads MACS	Miltenyi Biotec	130-048-701
Anti-Flk- 1 antibody	Abcam	ab24313
Anti-Ki67 antibody	CST	34330
Anti-PDGFRα antibody	Abcam	ab131591
Anti-Sca- 1 antibody	Invitrogen	710952
CD140a (PDGFRA) Monoclonal Antibody (APA5), FITC	eBioscience Invitrogen	11-1401-82
CD31 (PECAM-1) Monoclonal Antibody (390), APC	eBioscience Invitrogen	17-0311-82
CD34 Antibody, anti-mouse, FITC, REAfinity Clone REA383	Miltenyi Biotec	130-117-775
cell culture hood	JIANGSU SUJING GROUP CO.,LTD	SW-CJ-2FD
Centrifuge	CENCE	L530
CO₂ incubators	Thermofisher Scientific	4111
Confocal laser scanning microscope	Zeiss	zeiss 980
DMEM High Glucose Medium	ATCC	30-2002
EBM-2 culture medium	Lonza	CC-3162
FACSMelody	BD Biosciences
FACSMelody™ System	BD
Fetal bovine serum	Millipore	ES-009-C
FM-2 culture medium	ScienCell	2331
Fura-2/AM	Invitrogen	M1292
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488	Thermofisher Scientific	A32731
Leukemia inhibitory factor	Millipore	LIF2010
Microscope	Olympus	IX71
MiniMACS Starting Kit	Miltenyi Biotec	130-090-312
Penicillin-Streptomycin-Amphotericin B Solution	Beyotime	C0224
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)	BD Pharmingen	553141
Stereo Microscope	Olympus	SZX10
TILLvisION 4.0 program	T.I.L.L.Photonics GmbH	polychrome V
VWF Monoclonal Antibody (F8/86)	Thermofisher Scientific	MA5-14029
β-Mercaptoethanol	Thermofisher Scientific	21985023

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