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 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 vessels^2,3,4,5,6. 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 purity^{7,8,9,10,11,12}. 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 cells^13,14. 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).

1. Tissue block culture of adventitia from Aorta and mesenteric arteries

1. Resident CD34⁺ VW-SCs isolation
   1. Anesthetize two mice with 3% isoflurane inhalation (following institutionally approved protocols). Confirm adequate anesthesia by toe-pinch. Position the mouse supine with medical tape and spray 70% ethanol sanitizer to disinfect the fur of the mouse. Open the thoracic and abdominal cavities using sterile ophthalmic scissors and forceps to expose the heart and intestines. After dissecting the isolated aorta and mesenteric arteries, pin the aorta and mesenteric bed in a petri dish containing silicon elastomer and filled with a physiological salt solution. With another set of sterilized scissors and forceps, rapidly dissect off the fat around the aorta and mesenteric arteries.
   2. Transfer the dissected tissues to a 6 cm Petri dish containing 1% Penicillin-Streptomycin-Amphotericin B solution (see Table of Materials). After rinsing twice, transfer the arteries to a tissue culture hood under a stereo microscope.
   3. Cut the arteries longitudinally and use fine forceps to peel off the outer layer of the aorta and the first branch of mesenteric arteries. Transfer the outer layers to a 3.5 cm Petri dish containing 0.1-0.2 mL of culture medium (see Table of Materials). Cut the outer layers into tiny pieces (about 1 mm³).
   4. Transfer the tissue pieces into a gelatin-coated T25 flask, ensuring even distribution on the flask's bottom. Maintain a moderate distance between tissue pieces to facilitate cell crawling and growth. Place the flask vertically in an incubator (37 °C, 5% CO₂) for 3 h to allow tissue blocks to adhere to the flask's bottom.
   5. After 3 h, add 5 mL of DMEM high glucose growth medium to submerge the tissue blocks. Orient the T25 flask horizontally. Monitor cell migration around tissue blocks through a microscope at 3-day intervals.
      NOTE: The medium should contain 20% fetal bovine serum, 0.2% Leukemia Inhibitory Factor (LIF), 0.2 mM β-Mercaptoethanol, and 1% penicillin/streptomycin (see Table of Materials). Fetal bovine serum supports cell proliferation, while LIF and β-Mercaptoethanol impede cell differentiation^7,8.
   6. When cells in the T25 flask reach about 80% confluence, passage and culture them into one or two T75 flasks. Subculture the cells over five passages to ensure proper growth and health.
2. Magnetic separation of resident CD34⁺ stem cells
   1. When the cells in one or two T75 flasks reach 80% confluence, dissociate the cells with Trypsin, and resuspend them in 1 mL of sorting buffer (2 mM EDTA and 0.5% FBS). Determine the cell number (10⁷/T75 flask) and centrifuge the cell suspension at 300 x g for 5 min (at room temperature).
   2. Discard the supernatant completely and resuspend the cell pellet in 98 µL of sorting buffer per 10⁷ total cells. Add 2 µL CD34 antibody (see Table of Materials). Mix well and incubate for 30 min at 4°C in the dark with occasional shaking.
   3. Wash cells by adding 2 mL of sorting buffer and centrifuge at 300 x g for 10 min at room temperature.
   4. Remove the supernatant carefully and completely, and resuspend the cells in 80 µL of sorting buffer.
   5. Add 20 µL Anti-FITC MicroBeads (per 10⁷ total cells, see Table of Materials) into the buffer. Mix well by repetitive pipetting and incubate for 15 min at 4 °C in the dark with intermittent shaking.
   6. Wash the cells twice by adding 2 mL of buffer and centrifuge at 300 x g for 5 min to repellet cells. Discard the supernatant completely and resuspend the cells in 1 mL of buffer.
   7. Position one MS column within the magnetic field of an appropriate separator, ensuring that a collection tube (e.g., a 15 mL centrifuge tube) is placed beneath the MS column to collect the separated components. (see Table of Materials). Rinse the column with 500 µL buffer and wait until it has completely run through.
   8. Load the cell suspension into the column and collect the flow-through containing unlabeled cells into the 15 mL tube.
   9. Remove the column from the separator and place it on a new 15 mL centrifuge tube. Introduce 500 µL of buffer into the column, then promptly flush out the magnetically labeled cells by pushing the plunger into the column.
   10. To enhance the purity of CD34⁺ cells, the eluted fraction can undergo enrichment using a second MS column.Repeat the separation process as mentioned above. Assess the purity of the sorted cells via flow cytometry.
       ​NOTE: Typically, 10%-20% CD34⁺ cells are obtained, with around 70%-80% negative cells, and approximately 10% loss due to staining and centrifuge procedures.
   11. To further confirm the purity of vascular wall CD34⁺ stem cells, identify the sorted cells by flow cytometry. The present study showed a purity of more than 90%.

2. Cell identification by immunofluorescence

1. Seed cells on coverslips (diameter 8 mm) pre-coated with 0.04% gelatin.
2. When the cells reach about 60%-70% confluence, rinse two times with PBS and fix the cells with 4% paraformaldehyde for 10 min.
3. Wash cells with PBS for 3 min (3 times), and permeabilize the cells with 0.2% Triton X-100 (in PBS) for 5 min at room temperature.
4. Wash cells with PBS for 3 min (3 times), and block the non-specific binding of the antibodies with 5% donkey serum (in PBS) for 1 h.
5. Remove the blocking buffer by holding each coverslip on its edge with forceps and draining it onto a sheet of lint free wipe.
6. Incubate the cells in 100 times diluted primary antibodies (CD34, Flk-1, c-kit, Sca-1, Ki67, see Table of Materials) in 1% BSA (in PBS) overnight at 4 °C.
7. Decant the solution and wash the cells with PBS for 3 min (3 times). Incubate the cells with Alexa Fluor 488 labeled secondary antibody (1:200 in 1% BSA) (see Table of Materials) for 1 h at room temperature in the dark.
8. Perform DAPI counterstaining by diluting DAPI stock solution to 0.5 µg/mL in PBS and incubating cells for 5 min.
9. Rinse with PBS and seal the coverslips with antifade reagent. Capture images under the laser scanning confocal microscope.

3. Induced differentiation and characterization of CD34⁺ VW-SCs

1. Seed CD34⁺ stem cells at an appropriate density (40%-50% confluence) on coverslips and 6-well plates, respectively.
2. Induce endothelium cell-oriented differentiation from CD34⁺ cells by culturing cells in endothelial cell culture medium EBM-2 (see Table of Materials) for 1 week.
3. Induce fibroblast cell-oriented differentiation from CD34⁺ cells by culturing cells in fibroblast cell culture medium FM-2 (see Table of Materials) for 3 days.
4. Subject the undifferentiated and differentiated cells to immunofluorescence staining (as mentioned in step 2) at laser excitation of 488 nm and a 40x objective. Detect the expression of endothelial cell markers (CD31, VWF) and fibroblast cell markers (PDGFRα, Vimentin) (see Table of Materials).
5. Dissociate the cells with Trypsin and obtain the cell pellet by centrifugation (300 x g, 5 min, room temperature). Resuspend cells with 100 µL of sorting buffer. Preincubate the cell suspension with purified anti-mouse CD16/CD32 mAb (1 µg) (see Table of Materials) at 4 ˚C for 5 min.
6. Add 2 µL of PE Rat anti-Mouse CD34 (1:50), CD31 (PECAM-1) Monoclonal Antibody APC (2 µL, 1:50), and CD140a (PDGFRa) Monoclonal Antibody FITC (2 µL, 1:50) directly to preincubated cells in the presence of Mouse Fc Block and further incubate at 4 °C for 15 min.
7. Wash the cells twice by adding 2 mL of buffer and centrifuge at 300 x g for 5 min (room temperature) to repellet the cells. Discard the supernatant completely and resuspend the cells in 300 µL of buffer. Put flow tubes with stained cells into an icebox ready for Flow cytometry.

4. Detection of intracellular Ca²⁺ signaling in vascular CD34⁺ stem cells

1. Seed cells on coverslips 10 h before experiments at a density up to 70% confluence.
2. Take out the coverslips and affix them to the bottom of a custom chamber with any petroleum jelly.
3. Wash the coverslips once with PBS, replace with Fura-2/AM (5 µM) in Tyrode's solution (NaCl 137 mM, KCl 5.4 mM, MgCl₂ 1.2 mM, glucose 10 mM, HEPES 10 mM, CaCl₂ 2.4 mM) (see Table of Materials), and incubate cells for 30 min in the dark.
4. Remove the dye by treating with Tyrode's solution for 10 min to allow for fura-2/AM de-esterification.
5. Mount the chamber on the stage of an inverted fluorescence microscope equipped with a wide-field imaging system containing a monochromator and a CCD camera (see Table of Materials).
6. Open the main window, go to the Grab Settings dialog and camera page, and press the live button for live image display.
7. Choose the fluorescence image type, set the 340 nm/380 nm loop repeat times, image size, and camera exposure time at 380 nm and 340 nm to achieve optimal image brightness.
8. Take single snapshots, and draw several freehand lines to circle the cells with different colors and predefine Region of Interests (ROIs), indicating the background as ROI 0.
9. Re-edit an already embedded protocol in the workspace view and create a new workspace.
10. Execute the protocol, and the "Fluorescence 340 nm div Fluorescence 380 nm (Live Kinetic)" will display the online kinetic graph.
11. In the numerical view, a spreadsheet will be visible, presenting columns of gray-scale values and corresponding time information. Export the spreadsheet data by using the clipboard.
12. Calculate F340 nm/F380 nm indicating intracellular Ca²⁺ changes after subtracting the background fluorescence (ROI 0).

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...

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