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Isolation of Foreign Material-Free Endothelial Progenitor Cells Using CD31 Aptamer and Therapeutic Application for Ischemic Injury.

Yoon JW, Jang IH, Heo SC, Kwon YW, Choi EJ, Bae KH, Suh DS, Kim SC, Han S, Haam S, Jung J, Kim K, Ryu SH, Kim JH - PLoS ONE (2015)

Bottom Line: To utilize apatmers for isolation of EPCs, in the present study, we successfully generated aptamers that recognize human CD31, an endothelial cell marker.From the mixture of EPCs and 293FT cells, CD31 aptamers successfully isolated EPCs with 97.6% purity and 94.2% yield, comparable to those from antibody isolation.In this study, we demonstrated isolation of foreign material-free EPCs, which can be utilized as a universal protocol in preparation of cells for therapeutic transplantation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, Pusan National University, Yangsan 626-870, Republic of Korea.

ABSTRACT
Endothelial progenitor cells (EPCs) can be isolated from human bone marrow or peripheral blood and reportedly contribute to neovascularization. Aptamers are 40-120-mer nucleotides that bind to a specific target molecule, as antibodies do. To utilize apatmers for isolation of EPCs, in the present study, we successfully generated aptamers that recognize human CD31, an endothelial cell marker. CD31 aptamers bound to human umbilical cord blood-derived EPCs and showed specific interaction with human CD31, but not with mouse CD31. However, CD31 aptamers showed non-specific interaction with CD31-negative 293FT cells and addition of polyanionic competitor dextran sulfate eliminated non-specific interaction without affecting cell viability. From the mixture of EPCs and 293FT cells, CD31 aptamers successfully isolated EPCs with 97.6% purity and 94.2% yield, comparable to those from antibody isolation. In addition, isolated EPCs were decoupled from CD31 aptamers with a brief treatment of high concentration dextran sulfate. EPCs isolated with CD31 aptamers and subsequently decoupled from CD31 aptamers were functional and enhanced the restoration of blood flow when transplanted into a murine hindlimb ischemia model. In this study, we demonstrated isolation of foreign material-free EPCs, which can be utilized as a universal protocol in preparation of cells for therapeutic transplantation.

No MeSH data available.


Related in: MedlinePlus

CD31 aptamers specifically stain EPCs for visualization with fluorescence microscopy.(A) EPCs or 293FT cells were stained with CD31 antibodies (Alexa Fluor 488-labeled) and DAPI (blue). Images were taken by confocal microscope (Olympus FluoView FV1000). (B) EPCs or 293FT cells were stained with CD31 aptamers (AT-1, biotin-labeled, 400 nM) at 37°C for 1 h with or without 0.2 mM dextran sulfate, followed by staining with streptavidin-Alexa Fluor 488 and DAPI (blue). Images were taken by confocal microscope (Olympus FluoView FV1000). Scale bar = 40 μm (n = 3).
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pone.0131785.g003: CD31 aptamers specifically stain EPCs for visualization with fluorescence microscopy.(A) EPCs or 293FT cells were stained with CD31 antibodies (Alexa Fluor 488-labeled) and DAPI (blue). Images were taken by confocal microscope (Olympus FluoView FV1000). (B) EPCs or 293FT cells were stained with CD31 aptamers (AT-1, biotin-labeled, 400 nM) at 37°C for 1 h with or without 0.2 mM dextran sulfate, followed by staining with streptavidin-Alexa Fluor 488 and DAPI (blue). Images were taken by confocal microscope (Olympus FluoView FV1000). Scale bar = 40 μm (n = 3).

Mentions: We tested whether CD31 aptamers could be used in visualizing EPCs as antibodies in immunocytochemistry. When EPCs or 293FT cells were plated separately, CD31 antibodies specifically stained EPCs (Fig 3A). Following the immunocytochemistry protocol with aptamer binding buffer, CD31 aptamers were applied to EPCs or 293FT cells. Without dextran sulfate, CD31 aptamers showed strong interaction with EPCs (Fig 3B). Low level interaction with 293FT cells was also observed though low level interaction is weaker than that in flow cytometry analysis probably due to increased washing in immunocytochemistry protocol. However, addition of 0.2 mM dextran sulfate eliminated non-specific interaction of CD31 aptamers with 293FT cells but conserved the specific interaction with EPCs. These results demonstrate that CD31 aptamers can be used for visualizing EPCs with efficiency comparable to that of CD31 antibodies in immunocytochemistry.


Isolation of Foreign Material-Free Endothelial Progenitor Cells Using CD31 Aptamer and Therapeutic Application for Ischemic Injury.

Yoon JW, Jang IH, Heo SC, Kwon YW, Choi EJ, Bae KH, Suh DS, Kim SC, Han S, Haam S, Jung J, Kim K, Ryu SH, Kim JH - PLoS ONE (2015)

CD31 aptamers specifically stain EPCs for visualization with fluorescence microscopy.(A) EPCs or 293FT cells were stained with CD31 antibodies (Alexa Fluor 488-labeled) and DAPI (blue). Images were taken by confocal microscope (Olympus FluoView FV1000). (B) EPCs or 293FT cells were stained with CD31 aptamers (AT-1, biotin-labeled, 400 nM) at 37°C for 1 h with or without 0.2 mM dextran sulfate, followed by staining with streptavidin-Alexa Fluor 488 and DAPI (blue). Images were taken by confocal microscope (Olympus FluoView FV1000). Scale bar = 40 μm (n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493074&req=5

pone.0131785.g003: CD31 aptamers specifically stain EPCs for visualization with fluorescence microscopy.(A) EPCs or 293FT cells were stained with CD31 antibodies (Alexa Fluor 488-labeled) and DAPI (blue). Images were taken by confocal microscope (Olympus FluoView FV1000). (B) EPCs or 293FT cells were stained with CD31 aptamers (AT-1, biotin-labeled, 400 nM) at 37°C for 1 h with or without 0.2 mM dextran sulfate, followed by staining with streptavidin-Alexa Fluor 488 and DAPI (blue). Images were taken by confocal microscope (Olympus FluoView FV1000). Scale bar = 40 μm (n = 3).
Mentions: We tested whether CD31 aptamers could be used in visualizing EPCs as antibodies in immunocytochemistry. When EPCs or 293FT cells were plated separately, CD31 antibodies specifically stained EPCs (Fig 3A). Following the immunocytochemistry protocol with aptamer binding buffer, CD31 aptamers were applied to EPCs or 293FT cells. Without dextran sulfate, CD31 aptamers showed strong interaction with EPCs (Fig 3B). Low level interaction with 293FT cells was also observed though low level interaction is weaker than that in flow cytometry analysis probably due to increased washing in immunocytochemistry protocol. However, addition of 0.2 mM dextran sulfate eliminated non-specific interaction of CD31 aptamers with 293FT cells but conserved the specific interaction with EPCs. These results demonstrate that CD31 aptamers can be used for visualizing EPCs with efficiency comparable to that of CD31 antibodies in immunocytochemistry.

Bottom Line: To utilize apatmers for isolation of EPCs, in the present study, we successfully generated aptamers that recognize human CD31, an endothelial cell marker.From the mixture of EPCs and 293FT cells, CD31 aptamers successfully isolated EPCs with 97.6% purity and 94.2% yield, comparable to those from antibody isolation.In this study, we demonstrated isolation of foreign material-free EPCs, which can be utilized as a universal protocol in preparation of cells for therapeutic transplantation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, School of Medicine, Pusan National University, Yangsan 626-870, Republic of Korea.

ABSTRACT
Endothelial progenitor cells (EPCs) can be isolated from human bone marrow or peripheral blood and reportedly contribute to neovascularization. Aptamers are 40-120-mer nucleotides that bind to a specific target molecule, as antibodies do. To utilize apatmers for isolation of EPCs, in the present study, we successfully generated aptamers that recognize human CD31, an endothelial cell marker. CD31 aptamers bound to human umbilical cord blood-derived EPCs and showed specific interaction with human CD31, but not with mouse CD31. However, CD31 aptamers showed non-specific interaction with CD31-negative 293FT cells and addition of polyanionic competitor dextran sulfate eliminated non-specific interaction without affecting cell viability. From the mixture of EPCs and 293FT cells, CD31 aptamers successfully isolated EPCs with 97.6% purity and 94.2% yield, comparable to those from antibody isolation. In addition, isolated EPCs were decoupled from CD31 aptamers with a brief treatment of high concentration dextran sulfate. EPCs isolated with CD31 aptamers and subsequently decoupled from CD31 aptamers were functional and enhanced the restoration of blood flow when transplanted into a murine hindlimb ischemia model. In this study, we demonstrated isolation of foreign material-free EPCs, which can be utilized as a universal protocol in preparation of cells for therapeutic transplantation.

No MeSH data available.


Related in: MedlinePlus