murine aortal and mesenteric arteries isolation and culture to separate cd34+ stem cells 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.

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

まず、麻酔をかけたマウスを手術台に置きます。滅菌眼科用ハサミと鉗子を使用して、胸腔と腹腔を開きます。これにより、心臓と腸が露出します。腸間膜動脈と大動脈を別々に慎重に解剖します。腸間膜動脈をPBSですすぎ、1%ペニシリン-ストレプトマイシン-アンホテリシンB溶液を含む10センチメートルのシャーレに移します。.同様に、大動脈をすすぎ、同じ溶液を含む別のペトリ皿に移します。大動脈と腸間膜動脈の周りのすべての脂肪をすばやく取り除きます。2回すすいだ後、動脈を組織培養フード内の実体顕微鏡下に置きます。動脈に沿って慎重に縦方向に切り込みを入れます。次に、細い鉗子を使用して、大動脈の外層と腸間膜動脈の最初の枝をはがします。0.1〜0.2ミリリットルの培地が入った3.5cmのシャーレに外層を置き、約1立方ミリメートルの小片に切ります。ティッシュ片をゼラチンでコーティングされたT-25フラスコに移します。フラスコを摂氏37度のインキュベーターに垂直に置き、二酸化炭素を5%にします。3時間後、5ミリリットルのDMEM高グルコース増殖培地をフラスコに追加して、すべての組織ブロックを沈めます。

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 ...

医学

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

心血管研究のためのマウス大動脈および腸間膜動脈から単離された血管壁常在型CD34+ 幹細胞の磁気分離

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

心血管研究のための磁気的に単離されたCD34+ 幹細胞の特性評価

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

磁気的に分離された血管壁CD34+ 幹細胞における細胞内カルシウムシグナルの検出