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

혈관벽에서 murine CD34+ 줄기 세포의 분리 및 자기 분리

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.

저자 스포트라이트: Magnetic Bead Screening-Coupled Flow Cytometry를 사용한 혈관벽 거주자 쥐 CD34+ 줄기 세포의 향상된 분리 및 특성 분석

생쥐의 혈관벽 거주자 CD34+ 줄기 세포의 면역자기 분리

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.

심혈관 연구를 위한 자기적으로 분리된 CD34+ 줄기 세포의 특성 분석

쥐 대동맥 및 장간막 동맥에서 분리한 CD34+줄기세포를 특성화하기 위해 조직 블록 배양을 수행하고 자기 분리로 세포를 얻습니다. 분화 후 섬유아세포 마커 발현에서 내피 세포를 검출하려면 488나노미터의 레이저 여기로 40배 배율로 세포를 관찰하십시오. 세포를 트립시화 한 후 원심 분리로 펠릿화하고 100 마이크로 리터의 선별 완충액에 재현탁시킵니다.그런 다음 항 마우스 CD16 / CD32 단일 클론 항체로 섭씨 4도에서 5 분 동안 배양합니다. 그런 다음 면역 형광 염색을 위해 원하는 각 단클론 항체 2 마이크로리터를 추가하고 섭씨 4도에서 10분 동안 다시 배양합니다. 1 밀리리터의 완충액으로 세포를 두 번 씻으십시오.시연된 바와 같이 세포를 원심분리한 후, 상층액을 제거하고 세포를 300마이크로리터의 완충액에 재현탁시킵니다. 세포를 플로우 튜브로 옮기고 아이스 박스에 넣습니다. 유세포 분석을 실행하여 분화된 세포의 비율을 확인합니다.유세포 분석을 통해 분리된 CD34+혈관벽 줄기세포의 고순도를 확인했습니다. 세포 면역형광 염색 결과 분리된 CD34+줄기세포는 줄기세포 마커인 CD34, c-kit 및 Flk-1을 주로 발현하는 것으로 나타났습니다. 또한 유세포 분석을 통해 분리된 CD34+줄기세포가 in vitro에서 내피세포와 섬유아세포로 분화할 수 있는 능력이 있음을 확인했습니다.

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 ...

연구

의학

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

심혈관 연구를 위한 CD34+ 줄기 세포를 분리하기 위한 쥐 대동맥 및 장간막 동맥 분리 및 배양

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

심혈관 연구를 위해 쥐 대동맥 및 장간막 동맥에서 분리된 혈관벽 거주 CD34+ 줄기 세포의 자기 분리

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

자기적으로 격리된 혈관벽 CD34+ 줄기세포에서 세포내 칼슘 신호전달 검출