magnetic separation of vascular wall-resident cd34+ stem cells isolated from murine aortal and mesenteric arteries for cardiovascular studies

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

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&#39;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 &#39;quality control&#39; 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.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34+ Stem Cells Using Magnetic Bead Screening-Coupled Flow Cytometry

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

Resident vascular wall CD34+stem cells are increasingly recognized for their crucial role in regulating vascular remodeling post-injury. Establishing a stable and efficient method to culture functional CD34+cells is essential for further investigating the participating mechanisms under various physiological and pathological conditions. This technique combines magnetic bead screening and flow cytometry to purify the primary cultured resident CD34+stem cells.Then, the purified cells can be functionally identified through immunofluorescent staining and calcium imaging. Due to the phenotypical and functional heterogeneity of resident CD34+stem cells in the vascular nerve wall, it will be necessary to identify the specific subcellular type of resident vascular CD34+cells. We present a refined methodology for the culture, identification, and the functional assessment of vascular wall-resident CD34+stem cells.This novel approach encompasses magnetic sorting, flow cytometry, immunofluorescence staining, and calcium signaling measurement. By employing these techniques, our study establishes a solid foundation for the future comprehensive investigations into the function under regulation of recipient CD34+stem cells under both physiological and pathological conditions.

Magnetic Separation of Vascular Wall-Resident CD34+ Stem Cells Isolated from Murine Aortal and Mesenteric Arteries for Cardiovascular Studies

To begin, isolate and culture the outer layer of the aorta and the first branch of the mesenteric arteries isolated from the anesthetized mouse. Once the cells become 80%confluent, centrifuge the dissociated cell suspension at 300G for five minutes at room temperature, discard the supernatant completely and resuspend the cell pellet in 98 microliters of sorting buffer per 10 million total cells. After the addition of CD34 antibody, incubate the cells in the dark for 30 minutes at four degrees Celsius.To wash the cells, add two milliliters of sorting buffer to the cells, and centrifuge at 300G for 10 minutes. After discarding the supernatant, resuspend the cells in 80 microliters of sorting buffer. Then add 20 microliters of Anti-FITC MicroBeads to the cell suspension.After washing and centrifuging the cells as demonstrated previously, resuspend the cells in one milliliter of sorting buffer. Next, position a magnetic separation column within the magnetic field of a separator, and place a collection tube beneath the column. After rinsing with 500 microliters of buffer, load the cell suspension into the column and collect the flow through containing unlabeled cells in the collection tube.Remove the column from the separator and place it on a fresh 15 milliliter centrifuge tube. Introduce 500 microliters of sorting buffer into the column and push the plunger into the column to flush out the magnetically labeled cells.

Research

JoVE Journal

Medicine

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Murine Aortal and Mesenteric Arteries Isolation and Culture to Separate CD34+ Stem Cells for Cardiovascular Studies

Characterization of the Magnetically Isolated CD34+ Stem Cells for Cardiovascular Studies

Detection of Intracellular Calcium Signaling in the Magnetically Isolated Vascular Wall CD34+ Stem Cells