This work was funded by grants from National Natural Science Foundation of China (No. 82070502, 31972909, 32171099), the Sichuan Science and Technology Program of Sichuan Province (23NSFSC0576, 2022YFS0607). The authors would like to thank Qingbo Xu from Zhejiang University for help with the cell culture, and the authors acknowledge the scientific and technical assistance of the flow cytometry platform in Southwest Medical University.

This study was approved, and the animals were handled in accordance with the Guidelines for the Management and Use of Laboratory Animals in China. The research strictly adhered to the ethical requirements of animal experiments, with approval from the Animal Ethics Committee (Approval Number: SWMU2020664). Eight-week-old healthy C57BL/6 mice of either gender, weighing between 18-20 g, were utilized for the present study. The animals were housed at the Laboratory Animal Center of Southwest Medical University (SWMU).
1. Tissue block culture of adventitia from Aorta and mesenteric arteries
<ol>
	<li>Resident CD34+ VW-SCs isolation
	<ol>
		<li>Anesthetize two mice with 3% isoflurane inhalation (following institutionally approved protocols). Confirm adequate anesthesia by toe-pinch. Position the mouse supine with medical tape and spray 70% ethanol sanitizer to disinfect the fur of the mouse. Open the thoracic and abdominal cavities using sterile ophthalmic scissors and forceps to expose the heart and intestines. After dissecting the isolated aorta and mesenteric arteries, pin the aorta and mesenteric bed in a petri dish containing silicon elastomer and filled with a physiological salt solution. With another set of sterilized scissors and forceps, rapidly dissect off the fat around the aorta and mesenteric arteries.</li>
		<li>Transfer the dissected tissues to a 6 cm Petri dish containing 1% Penicillin-Streptomycin-Amphotericin B solution (see Table of Materials). After rinsing twice, transfer the arteries to a tissue culture hood under a stereo microscope.</li>
		<li>Cut the arteries longitudinally and use fine forceps to peel off the outer layer of the aorta and the first branch of mesenteric arteries. Transfer the outer layers to a 3.5 cm Petri dish containing 0.1-0.2 mL of culture medium (see Table of Materials). Cut the outer layers into tiny pieces (about 1 mm3).</li>
		<li>Transfer the tissue pieces into a gelatin-coated T25 flask, ensuring even distribution on the flask&#39;s bottom. Maintain a moderate distance between tissue pieces to facilitate cell crawling and growth. Place the flask vertically in an incubator (37 &#176;C, 5% CO2) for 3 h to allow tissue blocks to adhere to the flask&#39;s bottom.</li>
		<li>After 3 h, add 5 mL of DMEM high glucose growth medium to submerge the tissue blocks. Orient the T25 flask horizontally. Monitor cell migration around tissue blocks through a microscope at 3-day intervals. 
		NOTE: The medium should contain 20% fetal bovine serum, 0.2% Leukemia Inhibitory Factor (LIF), 0.2 mM &#946;-Mercaptoethanol, and 1% penicillin/streptomycin (see Table of Materials). Fetal bovine serum supports cell proliferation, while LIF and &#946;-Mercaptoethanol impede cell differentiation7,8.</li>
		<li>When cells in the T25 flask reach about 80% confluence, passage and culture them into one or two T75 flasks. Subculture the cells over five passages to ensure proper growth and health.</li>
	</ol>
	</li>
	<li>Magnetic separation of resident CD34+ stem cells
	<ol>
		<li>When the cells in one or two T75 flasks reach 80% confluence, dissociate the cells with Trypsin, and resuspend them in 1 mL of sorting buffer (2 mM EDTA and 0.5% FBS). Determine the cell number (107/T75 flask) and centrifuge the cell suspension at 300 x g for 5 min (at room temperature).</li>
		<li>Discard the supernatant completely and resuspend the cell pellet in 98 &#181;L of sorting buffer per 107 total cells. Add 2 &#181;L CD34 antibody (see Table of Materials). Mix well and incubate for 30 min at 4&#176;C in the dark with occasional shaking.</li>
		<li>Wash cells by adding 2 mL of sorting buffer and centrifuge at 300 x g for 10 min at room temperature.</li>
		<li>Remove the supernatant carefully and completely, and resuspend the cells in 80 &#181;L of sorting buffer.</li>
		<li>Add 20 &#181;L Anti-FITC MicroBeads (per 107 total cells, see Table of Materials) into the buffer. Mix well by repetitive pipetting and incubate for 15 min at 4 &#176;C in the dark with intermittent shaking.</li>
		<li>Wash the cells twice by adding 2 mL of buffer and centrifuge at 300 x g for 5 min to repellet cells. Discard the supernatant completely and resuspend the cells in 1 mL of buffer.</li>
		<li>Position one MS column within the magnetic field of an appropriate separator, ensuring that a collection tube (e.g., a 15 mL centrifuge tube) is placed beneath the MS column to collect the separated components. (see Table of Materials). Rinse the column with 500 &#181;L buffer and wait until it has completely run through.</li>
		<li>Load the cell suspension into the column and collect the flow-through containing unlabeled cells into the 15 mL tube.</li>
		<li>Remove the column from the separator and place it on a new 15 mL centrifuge tube. Introduce 500 &#181;L of buffer into the column, then promptly flush out the magnetically labeled cells by pushing the plunger into the column.</li>
		<li>To enhance the purity of CD34+ cells, the eluted fraction can undergo enrichment using a second MS column.Repeat the separation process as mentioned above. Assess the purity of the sorted cells via flow cytometry. 
		&#8203;NOTE: Typically, 10%-20% CD34+ cells are obtained, with around 70%-80% negative cells, and approximately 10% loss due to staining and centrifuge procedures.</li>
		<li>To further confirm the purity of vascular wall CD34+ stem cells, identify the sorted cells by flow cytometry. The present study showed a purity of more than 90%.</li>
	</ol>
	</li>
</ol>
2. Cell identification by immunofluorescence
<ol>
	<li>Seed cells on coverslips (diameter 8 mm) pre-coated with 0.04% gelatin.</li>
	<li>When the cells reach about 60%-70% confluence, rinse two times with PBS and fix the cells with 4% paraformaldehyde for 10 min.</li>
	<li>Wash cells with PBS for 3 min (3 times), and permeabilize the cells with 0.2% Triton X-100 (in PBS) for 5 min at room temperature.</li>
	<li>Wash cells with PBS for 3 min (3 times), and block the non-specific binding of the antibodies with 5% donkey serum (in PBS) for 1 h.</li>
	<li>Remove the blocking buffer by holding each coverslip on its edge with forceps and draining it onto a sheet of lint free wipe.</li>
	<li>Incubate the cells in 100 times diluted primary antibodies (CD34, Flk-1, c-kit, Sca-1, Ki67, see Table of Materials) in 1% BSA (in PBS) overnight at 4 &#176;C.</li>
	<li>Decant the solution and wash the cells with PBS for 3 min (3 times). Incubate the cells with Alexa Fluor 488 labeled secondary antibody (1:200 in 1% BSA) (see Table of Materials) for 1 h at room temperature in the dark.</li>
	<li>Perform DAPI counterstaining by diluting DAPI stock solution to 0.5 &#181;g/mL in PBS and incubating cells for 5 min.</li>
	<li>Rinse with PBS and seal the coverslips with antifade reagent. Capture images under the laser scanning confocal microscope.</li>
</ol>
3. Induced differentiation and characterization of CD34+ VW-SCs
<ol>
	<li>Seed CD34+ stem cells at an appropriate density (40%-50% confluence) on coverslips and 6-well plates, respectively.</li>
	<li>Induce endothelium cell-oriented differentiation from CD34+ cells by culturing cells in endothelial cell culture medium EBM-2 (see Table of Materials) for 1 week.</li>
	<li>Induce fibroblast cell-oriented differentiation from CD34+ cells by culturing cells in fibroblast cell culture medium FM-2 (see Table of Materials) for 3 days.</li>
	<li>Subject the undifferentiated and differentiated cells to immunofluorescence staining (as mentioned in step 2) at laser excitation&#160;of 488 nm and a 40x objective. Detect the expression of endothelial cell markers (CD31, VWF) and fibroblast cell markers (PDGFR&#945;, Vimentin) (see Table of Materials).</li>
	<li>Dissociate the cells with Trypsin and obtain the cell pellet by centrifugation (300 x g, 5 min, room temperature). Resuspend cells with 100 &#181;L of sorting buffer. Preincubate the cell suspension with purified anti-mouse CD16/CD32 mAb (1 &#181;g) (see Table of Materials) at 4 &#730;C for 5 min.</li>
	<li>Add 2 &#181;L of PE Rat anti-Mouse CD34 (1:50), CD31 (PECAM-1) Monoclonal Antibody APC (2 &#181;L, 1:50), and CD140a (PDGFRa) Monoclonal Antibody FITC (2 &#181;L, 1:50) directly to preincubated cells in the presence of Mouse Fc Block and further incubate at 4 &#176;C for 15 min.</li>
	<li>Wash the cells twice by adding 2 mL of buffer and centrifuge at 300 x g for 5 min (room temperature) to repellet the cells. Discard the supernatant completely and resuspend the cells in 300 &#181;L of buffer. Put flow tubes with stained cells into an icebox ready for Flow cytometry.</li>
</ol>
4. Detection of intracellular Ca2+ signaling in vascular CD34+ stem cells
<ol>
	<li>Seed cells on coverslips 10 h before experiments at a density up to 70% confluence.</li>
	<li>Take out the coverslips and affix them to the bottom of a custom chamber with any petroleum jelly.</li>
	<li>Wash the coverslips once with PBS, replace with Fura-2/AM (5 &#181;M) in Tyrode&#39;s solution (NaCl 137 mM, KCl 5.4 mM, MgCl2 1.2 mM, glucose 10 mM, HEPES 10 mM, CaCl2 2.4 mM) (see Table of Materials), and incubate cells for 30 min in the dark.</li>
	<li>Remove the dye by treating with Tyrode&#39;s solution for 10 min to allow for fura-2/AM de-esterification.</li>
	<li>Mount the chamber on the stage of an inverted fluorescence microscope equipped with a wide-field imaging system containing a monochromator and a CCD camera (see Table of Materials).</li>
	<li>Open the main window, go to the Grab Settings dialog and camera page, and press the live button for live image display.</li>
	<li>Choose the fluorescence image type, set the 340 nm/380 nm loop repeat times, image size, and camera exposure time at 380 nm and 340 nm to achieve optimal image brightness.</li>
	<li>Take single snapshots, and draw several freehand lines to circle the cells with different colors and predefine Region of Interests (ROIs), indicating the background as ROI 0.</li>
	<li>Re-edit an already embedded protocol in the workspace view and create a new workspace.</li>
	<li>Execute the protocol, and the &#34;Fluorescence 340 nm div Fluorescence 380 nm (Live Kinetic)&#34; will display the online kinetic graph.</li>
	<li>In the numerical view, a spreadsheet will be visible, presenting columns of gray-scale values and corresponding time information. Export the spreadsheet data by using the clipboard.</li>
	<li>Calculate F340 nm/F380 nm indicating intracellular Ca2+ changes after subtracting the background fluorescence (ROI 0).</li>
</ol>

Isolation and Magnetic Separation of Murine CD34&lt;sup&gt;+&lt;/sup&gt; Stem Cells from the Vascular Wall

<ol>
	<li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundant+progenitor+cells+in+the+adventitia+contribute+to+atherosclerosis+of+vein+grafts+in+ApoE-deficient+mice.">Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice.</a> J Clin Invest. 113 (9), 1258-1265 (2004).</li><li>Jiang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonbone+marrow+CD34++cells+are+crucial+for+endothelial+repair+of+the+injured+artery.">Nonbone marrow CD34+ cells are crucial for endothelial repair of the injured artery.</a> Circ Res. 129 (8), e146-e165 (2021).</li><li>Tamma, R., Ruggieri, S., Annese, T., Ribatti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+wall+as+a+source+of+stem+cells+able+to+differentiate+into+endothelial+cells.">Vascular wall as a source of stem cells able to differentiate into endothelial cells.</a> Adv Exp Med Biol. 1237, 29-36 (2020).</li><li>Patel, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+definition+of+progenitors+versus+mature+endothelial+cells+reveals+key+soxF-dependent+differentiation+process.">Functional definition of progenitors versus mature endothelial cells reveals key soxF-dependent differentiation process.</a> Circulation. 135 (8), 786-805 (2017).</li><li>Zhang, L., Issa Bhaloo, S., Chen, T., Zhou, B., Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+stem+cells+in+vascular+remodeling.">Roles of stem cells in vascular remodeling.</a> Chin J Cell Biol. 43 (7), 1352-1361 (2021).</li><li>Zhang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+resident+stem+cells+in+vessel+formation+and+arteriosclerosis.">Role of resident stem cells in vessel formation and arteriosclerosis.</a> Circ Res. 122 (11), 1608-1624 (2018).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+estrogen+on+growth+and+smooth+muscle+differentiation+of+vascular+wall-resident+CD34++stem/progenitor+cells.">Effects of estrogen on growth and smooth muscle differentiation of vascular wall-resident CD34+ stem/progenitor cells.</a> Atherosclerosis. 240 (2), 453-461 (2015).</li><li>Tang, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+vascular+wall-resident+CD34++stem/progenitor+cells.">Isolation and culture of vascular wall-resident CD34+ stem/progenitor cells.</a> Cardiol Plus. 4 (4), 116-120 (2019).</li><li>Sukumaran, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+regulates+autophagy+and+apoptosis.">Calcium signaling regulates autophagy and apoptosis.</a> Cells. 10 (8), 2125 (2021).</li><li>van der Sanden, B., Dhobb, M., Berger, F., Wion, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+stem+cell+culture.">Optimizing stem cell culture.</a> J Cell Biochem. 111 (4), 801-807 (2010).</li><li>Rotmans, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo,+cell+seeding+with+anti-CD34+antibodies+successfully+accelerates+endothelialization+but+stimulates+intimal+hyperplasia+in+porcine+arteriovenous+expanded+polytetrafluoroethylene+grafts.">In vivo, cell seeding with anti-CD34 antibodies successfully accelerates endothelialization but stimulates intimal hyperplasia in porcine arteriovenous expanded polytetrafluoroethylene grafts.</a> Circulation. 112 (1), 12-18 (2005).</li><li>Qu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+serum+amyloid+A1+in+the+adipogenic+differentiation+of+human+adipose-derived+stem+cells+basing+on+single-cell+RNA+sequencing+analysis.">The role of serum amyloid A1 in the adipogenic differentiation of human adipose-derived stem cells basing on single-cell RNA sequencing analysis.</a> Stem Cell Res Ther. 13 (1), 187 (2022).</li><li>Flynn, J., Gorry, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry+Analysis+to+Identify+Human+CD8++T+Cells.">Flow Cytometry Analysis to Identify Human CD8+ T Cells.</a> Methods Mol Biol. 2048, 1-13 (2019).</li><li>Bacon, K., Lavoie, A., Rao, B. M., Daniele, M., Menegatti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Past,+present,+and+future+of+affinity-based+cell+separation+technologies.">Past, present, and future of affinity-based cell separation technologies.</a> Acta Biomater. 112, 29-51 (2020).</li><li>Ma, H. G., Liu, H. Q., Liu, S. D., Tang, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+and+identification+of+rat+glomerular+microvascular+endothelial+cells.">Primary culture and identification of rat glomerular microvascular endothelial cells.</a> Acta Physiol Sin. 73 (6), 926-930 (2021).</li><li>Liu, W. H., Wang, P., Yang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+identification+of+rats+hair+follicle+neural+crest+stem+cells.">Isolation, culture and identification of rats hair follicle neural crest stem cells.</a> Chin J Neuroanat. 35 (2), 207-211 (2019).</li><li>Kumar, P., Garg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+approaches+to+obtain+medulloblastoma+stem+cells+from+bulk+cultures.">Flow cytometry approaches to obtain medulloblastoma stem cells from bulk cultures.</a> Methods Mol Biol. 2423, 87-94 (2022).</li><li>Haroon, M. M., Vemula, P. K., Palakodeti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+analysis+of+planarian+stem+cells+using+DNA+and+mitochondrial+dyes.">Flow cytometry analysis of planarian stem cells using DNA and mitochondrial dyes.</a> Bio Protoc. 12 (2), e4299 (2022).</li><li>Catchpole, T., Nguyen, T. D., Gilfoyle, A., Csaky, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+profile+of+circulating+vascular+progenitor+cells+in+human+neovascular+age-related+macular+degeneration.">A profile of circulating vascular progenitor cells in human neovascular age-related macular degeneration.</a> PLOS One. 15 (2), e0229504 (2020).</li><li>Wang, G., Yu, G., Wang, D., Guo, S., Shan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+purity+and+vitality+of+natural+killer+cells+with+different+isolation+kits.">Comparison of the purity and vitality of natural killer cells with different isolation kits.</a> Exp Ther Med. 13 (5), 1875-1883 (2017).</li><li>Moore, D. K., Motaung, B., du Plessis, N., Shabangu, A. N., Loxton, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consortium+SI.+Isolation+of+B-cells+using+Miltenyi+MACS+bead+isolation+kits.">Consortium SI. Isolation of B-cells using Miltenyi MACS bead isolation kits.</a> PLOS One. 14 (3), e0213832 (2019).</li><li>Jiang, L. H., Mousawi, F., Yang, X., Roger, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP-induced+Ca2+-signalling+mechanisms+in+the+regulation+of+mesenchymal+stem+cell+migration.">ATP-induced Ca2+-signalling mechanisms in the regulation of mesenchymal stem cell migration.</a> Cell Mol Life Sci. 74 (20), 3697-3710 (2017).</li><li>Ong, H. L., Subedi, K. P., Son, G. Y., Liu, X., Ambudkar, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+store-operated+calcium+entry+to+modulate+Ca2+-dependent+physiological+processes.">Tuning store-operated calcium entry to modulate Ca2+-dependent physiological processes.</a> Biochim Biophys Acta Mol Cell Res. 1866 (7), 1037-1045 (2019).</li><li>Garcia-Carlos, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin+II,+ATP+and+high+extracellular+potassium+induced+intracellular+calcium+responses+in+primary+rat+brain+endothelial+cell+cultures.">Angiotensin II, ATP and high extracellular potassium induced intracellular calcium responses in primary rat brain endothelial cell cultures.</a> Cell Biochem Funct. 39 (5), 688-698 (2021).</li><li>Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caffeine+as+a+tool+to+investigate+sarcoplasmic+reticulum+and+intracellular+calcium+dynamics+in+human+skeletal+muscles.">Caffeine as a tool to investigate sarcoplasmic reticulum and intracellular calcium dynamics in human skeletal muscles.</a> J Muscle Res Cell Motil. 42 (2), 281-289 (2021).</li></ol>

The authors have no conflicts of interest to disclose.

This study provides a quick and convenient method for obtaining functional CD34+ VW-SCs from the aorta and mesenteric arteries of mice. CD34+ VW-SCs obtained by this method have proliferative activity and multidirectional differentiation properties. Triphosphate inositol 1,4,5-trisphosphate receptors (IP3Rs), ryanodine receptors (RyRs), and store-operated calcium channels mediate Ca2+ release and entry in CD34+ VW-SCs. The establishment of this technique will lay the...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2% gelatin solution</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31 antibody</td>
			<td>R&#38;D</td>
			<td>AF3628</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD34 antibody</td>
			<td>Abcam</td>
			<td>ab81289</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-c-kit antibody</td>
			<td>CST</td>
			<td>77522</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-FITC MicroBeads</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-701&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-FITC MicroBeads MACS</td>
			<td>Miltenyi Biotec</td>
			<td>130-048-701</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Flk- 1 antibody</td>
			<td>Abcam</td>
			<td>ab24313</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Ki67 antibody</td>
			<td>CST</td>
			<td>34330</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PDGFR&#945; antibody</td>
			<td>Abcam</td>
			<td>ab131591</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Sca- 1 antibody</td>
			<td>Invitrogen</td>
			<td>710952</td>
			<td></td>
		</tr>
		<tr>
			<td>CD140a (PDGFRA) Monoclonal Antibody (APA5), FITC</td>
			<td>eBioscience &#160;Invitrogen</td>
			<td>11-1401-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 (PECAM-1) Monoclonal Antibody (390), APC</td>
			<td>eBioscience&#160; Invitrogen</td>
			<td>17-0311-82</td>
			<td></td>
		</tr>
		<tr>
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			<td>130-117-775</td>
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			<td>JIANGSU SUJING GROUP CO.,LTD&#160;</td>
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			<td>A32731</td>
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			<td>Millipore</td>
			<td>LIF2010</td>
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			<td>Olympus</td>
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			<td>Miltenyi Biotec</td>
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			<td>Beyotime</td>
			<td>C0224</td>
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			<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
			<td>BD Pharmingen</td>
			<td>553141</td>
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			<td>Olympus</td>
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			<td>TILLvisION 4.0 program&#160;</td>
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			<td>polychrome V&#160;</td>
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			<td>Thermofisher Scientific</td>
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			<td>Thermofisher Scientific</td>
			<td>21985023</td>
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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 and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

Author Spotlight: Enhanced Isolation and Characterization of Vascular Wall Resident Murine CD34&lt;sup&gt;+&lt;/sup&gt; 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.

Isolation and purification of CD34+ VW-SCs 
High purity of CD34+ VW-SCs is obtained from the adventitia of the mouse aortic and mesenteric artery by tissue attachment and magnetic microbead sorting. The percentage of CD34+ cells in the vessel wall is generally 10%-30%. Flow cytometry confirms that the purity of CD34+ cells obtained by magnetic bead sorting is more than 90% (Figure 1A). Cellular immunofluorescence staining show...

Watch this Scientific Journal Video about Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice at JoVE.com

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

immunomagnetic-isolation-vascular-wall-resident-cd34-stem-cells-from

Research

JoVE Journal

Medicine

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

To begin, place an anesthetized mouse on the operating table. Using sterile ophthalmic scissors and forceps, open the thoracic and abdominal cavities. This will expose the heart and the intestines.Carefully dissect the mesenteric arteries and the aorta separately. Rinse the mesenteric arteries in PBS and transfer them to a 10-centimeter Petri dish containing 1%penicillin-streptomycin-amphotericin B solution. Similarly, rinse the aorta and transfer it to another Petri dish containing the same solution.Quickly remove all the fat around the aorta and the mesenteric arteries. After rinsing twice, place the arteries under a stereo microscope in a tissue culture hood. Carefully make a longitudinal cut along the arteries.Then using fine forceps, peel off the outer layer of the aorta and the first branch of the mesenteric arteries. Place the outer layer in a 3.5 centimeter Petri dish containing 0.1 to 0.2 milliliters of culture medium, and chop it into tiny pieces of about one cubic millimeter. Transfer the tissue pieces into a gelatin-coated T-25 flask.Place the flask vertically in an incubator at 37 degree Celsius with 5%carbon dioxide. After three hours, add five milliliters of DMEM high glucose growth medium to the flask to submerge all the tissue blocks.

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

To begin, isolate and culture the outer layer of the aorta and the first branch of the mesenteric arteries isolated from the anesthetized mouse. Once the cells become 80%confluent, centrifuge the dissociated cell suspension at 300G for five minutes at room temperature, discard the supernatant completely and resuspend the cell pellet in 98 microliters of sorting buffer per 10 million total cells. After the addition of CD34 antibody, incubate the cells in the dark for 30 minutes at four degrees Celsius.To wash the cells, add two milliliters of sorting buffer to the cells, and centrifuge at 300G for 10 minutes. After discarding the supernatant, resuspend the cells in 80 microliters of sorting buffer. Then add 20 microliters of Anti-FITC MicroBeads to the cell suspension.After washing and centrifuging the cells as demonstrated previously, resuspend the cells in one milliliter of sorting buffer. Next, position a magnetic separation column within the magnetic field of a separator, and place a collection tube beneath the column. After rinsing with 500 microliters of buffer, load the cell suspension into the column and collect the flow through containing unlabeled cells in the collection tube.Remove the column from the separator and place it on a fresh 15 milliliter centrifuge tube. Introduce 500 microliters of sorting buffer into the column and push the plunger into the column to flush out the magnetically labeled cells.

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

To characterize the CD34+stem cells isolated from the murine aorta and mesenteric arteries, perform tissue block culture and obtain the cells by magnetic separation. To detect the endothelial cell in fibroblast cell marker expression after differentiation, observe the cells at 40x magnification with laser excitations of 488 nanometers. After trypsinizing the cells, pellet them by centrifugation and resuspend in 100 microliters of sorting buffer.Then incubate them with anti-mouse CD16/CD32 monoclonal antibodies at four degrees Celsius for five minutes. Then add two microliters of each desired monoclonal antibody for immunofluorescent staining and incubate again at 4 degrees Celsius for 10 minutes. Wash the cells twice with one milliliter of buffer.After centrifuging the cells as demonstrated, remove the supernatant and resuspend the cells in 300 microliters of buffer. Transfer the cells to the flow tubes and place them in an ice box. Run flow cytometry to check the percentage of differentiated cells.Flow cytometric evaluation confirmed the high purity of isolated CD34+vascular wall stem cells. Cellular immunofluorescent staining showed that the isolated CD34+stem cells predominantly expressed stem cell markers, CD34, c-kit, and Flk-1. Flow cytometry also confirmed that the isolated CD34+stem cells possessed the ability to differentiate into endothelial cells as well as fibroblasts in vitro.

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

To begin, isolate the resident vascular wall CD34-positive stem cells from murine models. Obtain pure cells by magnetic activated cell sorting and characterize them. For detecting calcium signaling, obtain a coverslip pre-seeded with the cells and wash it once with PBS.Then incubate the cells with Fura 2-AM and Tyrode solution for 30 minutes in the dark. To remove the dye, incubate the cells with Tyrode solution for 10 minutes. Then mount the chamber on the stage of an inverted fluorescence microscope, open the main window and go to the grab settings dialogue and camera page.Press the live button for the live image display. To acquire images, draw several freehand lines to circle the cells with different colors and predefine the regions of interest. Fluorescence microscopy demonstrated that ATP administration increased the intracellular calcium levels in the isolated vascular wall CD34-positive stem cells.

418 Views.  Southwest Medical University.  This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

Isolation and Magnetic Separation of Murine CD34+ Stem Cells from the Vascular Wall

Summary