characterization of the magnetically isolated cd34+ stem cells for cardiovascular studies

Isolierung und magnetische Trennung von murinen CD34+ -Stammzellen aus der Gefäßwand

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.

Autor im Rampenlicht: Verbesserte Isolierung und Charakterisierung von in der Gefäßwand ansässigen murinen CD34+ -Stammzellen mittels magnetischer Bead-Screening-gekoppelter Durchflusszytom

Immunmagnetische Isolierung von in der Gefäßwand ansässigen CD34+ -Stammzellen aus Mäusen

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.

Charakterisierung der magnetisch isolierten CD34+ Stammzellen für kardiovaskuläre Studien

Um die CD34+-Stammzellen zu charakterisieren, die aus der murinen Aorta und den mesenterialen Arterien isoliert wurden, führen Sie eine Gewebeblockkultur durch und gewinnen Sie die Zellen durch magnetische Trennung. Um die Endothelzelle in der Expression von Fibroblastenzellmarkern nach der Differenzierung nachzuweisen, werden die Zellen bei 40-facher Vergrößerung mit Laseranregungen von 488 Nanometern beobachtet. Nach der Trypsinisierung der Zellen werden sie durch Zentrifugation pelletiert und in 100 Mikrolitern Sortierpuffer resuspendiert.Dann inkubieren Sie sie fünf Minuten lang mit monoklonalen Anti-Maus-CD16/CD32-Antikörpern bei vier Grad Celsius. Fügen Sie dann zwei Mikroliter jedes gewünschten monoklonalen Antikörpers für die Immunfluoreszenzfärbung hinzu und inkubieren Sie erneut 10 Minuten lang bei 4 Grad Celsius. Waschen Sie die Zellen zweimal mit einem Milliliter Puffer.Nachdem Sie die Zellen wie gezeigt zentrifugiert haben, entfernen Sie den Überstand und resuspendieren Sie die Zellen in 300 Mikrolitern Puffer. Übertragen Sie die Zellen in die Durchflussrohre und legen Sie sie in eine Eisbox. Führen Sie eine Durchflusszytometrie durch, um den Prozentsatz der differenzierten Zellen zu überprüfen.Die durchflusszytometrische Auswertung bestätigte die hohe Reinheit der isolierten CD34+Gefäßwand-Stammzellen. Die zelluläre Immunfluoreszenzfärbung zeigte, dass die isolierten CD34+-Stammzellen überwiegend die Stammzellmarker CD34, c-kit und Flk-1 exprimierten. Die Durchflusszytometrie bestätigte auch, dass die isolierten CD34+-Stammzellen die Fähigkeit besaßen, sich sowohl in Endothelzellen als auch in Fibroblasten in vitro zu differenzieren.

Research

JoVE Journal

Medicine

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

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

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

Forschung

Medizin

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

Isolierung und Kultivierung von Aorten- und Mesenterialarterien von Mäusen zur Trennung von CD34+ -Stammzellen für kardiovaskuläre Studien

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

Magnetische Trennung von in der Gefäßwand ansässigen CD34+ -Stammzellen, die aus murinen Aorten- und Mesenterialarterien für kardiovaskuläre Studien isoliert wurden

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

Nachweis des intrazellulären Calcium-Signalwegs in den magnetisch isolierten CD34+ -Stammzellen der Gefäßwand