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

Murin CD34+ Kök Hücrelerinin Damar Duvarından İzolasyonu ve Manyetik Olarak Ayrılması

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.

Yazar Spot Işığı: Manyetik Boncuk Tarama-Birleştirilmiş Akış Sitometrisi Kullanılarak Vasküler Duvarda Yerleşik Murin CD34+ Kök Hücrelerinin Gelişmiş İzolasyonu ve Karakterizasyonu

Farelerden Vasküler Duvarda Yerleşik CD34+ Kök Hücrelerin İmmünomanyetik İzolasyonu

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.

Kardiyovasküler Çalışmalar için Manyetik Olarak İzole Edilmiş CD34+ Kök Hücrelerin Karakterizasyonu

Murin aort ve mezenterik arterlerden izole edilen CD34+kök hücreleri karakterize etmek için doku blok kültürü yapılır ve manyetik ayırma ile hücreler elde edilir. Farklılaşmadan sonra fibroblast hücre işaretleyici ekspresyonunda endotel hücresini tespit etmek için, hücreleri 488 nanometrelik lazer uyarımları ile 40x büyütmede gözlemleyin. Hücreleri tripsinledikten sonra, santrifüjleme ile pelet haline getirin ve 100 mikrolitre ayırma tamponunda yeniden süspanse edin.Daha sonra onları fare karşıtı CD16 / CD32 monoklonal antikorları ile dört santigrat derecede beş dakika boyunca inkübe edin. Daha sonra immünofloresan boyama için istenen her monoklonal antikorun iki mikrolitresini ekleyin ve 10 dakika boyunca 4 santigrat derecede tekrar inkübe edin. Hücreleri bir mililitre tamponla iki kez yıkayın.Hücreleri gösterildiği gibi santrifüjledikten sonra, süpernatanı çıkarın ve hücreleri 300 mikrolitre tamponda yeniden süspanse edin. Hücreleri akış tüplerine aktarın ve bir buz kutusuna yerleştirin. Farklılaşmış hücrelerin yüzdesini kontrol etmek için akış sitometrisini çalıştırın.Flow sitometrik değerlendirme, izole CD34 + vasküler duvar kök hücrelerinin yüksek saflığını doğruladı. Hücresel immünofloresan boyama, izole edilen CD34 + kök hücrelerin ağırlıklı olarak kök hücre belirteçlerini, CD34, c-kit ve Flk-1'i eksprese ettiğini gösterdi. Akış sitometrisi ayrıca izole edilen CD34 + kök hücrelerin in vitro olarak fibroblastların yanı sıra endotel hücrelerine farklılaşma yeteneğine sahip olduğunu doğruladı.

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 ...

Tıp

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

Kardiyovasküler Çalışmalar için Murin Aortal ve Mezenterik Arterlerin İzolasyonu ve CD34+ Kök Hücrelerini Ayırmak İçin Kültür

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

Kardiyovasküler Çalışmalar için Murin Aort ve Mezenterik Arterlerden İzole Edilen Vasküler Duvarda Yerleşik CD34+ Kök Hücrelerin Manyetik Olarak Ayrılması

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

Manyetik Olarak İzole Edilmiş Vasküler Duvar CD34+ Kök Hücrelerinde Hücre İçi Kalsiyum Sinyalizasyonunun Tespiti