detection of intracellular calcium signaling in the magnetically isolated vascular wall cd34+ stem cells

संवहनी दीवार से murine CD34+ स्टेम सेल का अलगाव और चुंबकीय पृथक्करण

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.

लेखक स्पॉटलाइट: संवहनी दीवार निवासी Murine CD34 + स्टेम सेल के उन्नत अलगाव और लक्षण वर्णन चुंबकीय मनका स्क्रीनिंग-युग्मित प्रवाह साइटोमेट्री का उपयोग कर

चूहों से संवहनी दीवार-निवासी CD34+ स्टेम सेल का इम्यूनोमैग्नेटिक अलगाव

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.

चुंबकीय रूप से पृथक संवहनी दीवार CD34+ स्टेम सेल में इंट्रासेल्युलर कैल्शियम सिग्नलिंग का पता लगाना

शुरू करने के लिए, murine मॉडल से निवासी संवहनी दीवार CD34 पॉजिटिव स्टेम कोशिकाओं को अलग. चुंबकीय सक्रिय सेल छँटाई द्वारा शुद्ध कोशिकाओं को प्राप्त करें और उन्हें चिह्नित करें। कैल्शियम सिग्नलिंग का पता लगाने के लिए, कोशिकाओं के साथ पूर्व-वरीयता प्राप्त एक कवरस्लिप प्राप्त करें और इसे पीबीएस के साथ एक बार धो लें।फिर अंधेरे में 30 मिनट के लिए फ्यूरा 2-एएम और टायरोड समाधान के साथ कोशिकाओं को इनक्यूबेट करें। डाई को हटाने के लिए, 10 मिनट के लिए टायरोड समाधान के साथ कोशिकाओं को सेते हैं। फिर एक औंधा प्रतिदीप्ति माइक्रोस्कोप के मंच पर चैम्बर माउंट, मुख्य खिड़की खोलने के लिए और हड़पने सेटिंग्स संवाद और कैमरा पृष्ठ पर जाना.लाइव छवि प्रदर्शन के लिए लाइव बटन दबाएं। छवियों को प्राप्त करने के लिए, विभिन्न रंगों के साथ कोशिकाओं को सर्कल करने और ब्याज के क्षेत्रों को पूर्वनिर्धारित करने के लिए कई फ्रीहैंड लाइनें खींचें। प्रतिदीप्ति माइक्रोस्कोपी ने प्रदर्शित किया कि एटीपी प्रशासन ने पृथक संवहनी दीवार सीडी 34-पॉजिटिव स्टेम कोशिकाओं में इंट्रासेल्युलर कैल्शियम के स्तर में वृद्धि की।

Research

JoVE Journal

Medicine

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

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 ...

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

हृदय अध्ययन के लिए CD34+ स्टेम कोशिकाओं को अलग करने के लिए Murine महाधमनी और मेसेंटेरिक धमनियों अलगाव और संस्कृति

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

कार्डियोवास्कुलर अध्ययन के लिए संवहनी दीवार-निवासी CD34+ स्टेम सेल का चुंबकीय पृथक्करण Murine महाधमनी और मेसेंटेरिक धमनियों से पृथक

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

हृदय अध्ययन के लिए चुंबकीय रूप से पृथक CD34+ स्टेम कोशिकाओं की विशेषता