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

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 int....

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).

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.......

Materials

Name	Company	Catalog Number	Comments
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