murine aortal and mesenteric arteries isolation and culture to separate cd34+ stem cells for cardiovascular studies

Isolering og magnetisk adskillelse af murine CD34+ stamceller fra karvæggen

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.

Forfatter Spotlight: Forbedret isolering og karakterisering af vaskulære væg bosiddende mus CD34+ stamceller ved hjælp af magnetisk perlescreening-koblet flowcytometri

Immunmagnetisk isolering af de vaskulære væg-residente CD34+ -stamceller fra mus

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.

Isolering og dyrkning af murine aortale og mesenteriske arterier til adskillelse af CD34+ -stamceller til kardiovaskulære undersøgelser

For at begynde skal du placere en bedøvet mus på operationsbordet. Brug sterile oftalmiske sakse og pincet til at åbne bryst- og abdominalhulen. Dette vil udsætte hjertet og tarmene.Dissekeres forsigtigt mesenterialarterierne og aorta separat. Skyl de mesenteriske arterier i PBS og overfør dem til en 10 centimeter petriskål indeholdende 1% penicillin-streptomycin-amphotericin B-opløsning. På samme måde skylles aorta og overføres til en anden petriskål, der indeholder den samme opløsning.Fjern hurtigt alt fedt omkring aorta og mesenterialarterierne. Efter skylning to gange skal du placere arterierne under et stereomikroskop i en vævskulturhætte. Lav forsigtigt et langsgående snit langs arterierne.Brug derefter en fin pincet til at skrælle det ydre lag af aorta og den første gren af mesenterialarterierne af. Læg det ydre lag i en 3,5 centimeter petriskål indeholdende 0,1 til 0,2 ml dyrkningsmedium, og hak det i små stykker på cirka en kubikmillimeter. Overfør vævsstykkerne til en gelatinebelagt T-25-kolbe.Kolben anbringes lodret i en inkubator ved 37 grader Celsius med 5% kuldioxid. Efter tre timer tilsættes fem milliliter DMEM højt glukosevækstmedium til kolben for at nedsænke alle vævsblokke.

Research

JoVE Journal

Medicine

Murine Aortal and Mesenteric Arteries Isolation and Culture to Separate 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 ...

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

Magnetisk adskillelse af vaskulære væg-residente CD34+ -stamceller isoleret fra murine aortale og mesenteriske arterier til kardiovaskulære undersøgelser

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

Karakterisering af de magnetisk isolerede CD34+ stamceller til kardiovaskulære studier

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

Påvisning af intracellulær calciumsignalering i de magnetisk isolerede vaskulære CD34+- stamceller