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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

Dextran sulfate effectively reduces the non-specific interaction of CD31 aptamers.(A) 293FT cells were incubated with various concentrations (0, 0.2, 2, 20, and 200 nM) of CD31 aptamer clone 1 (AT-1, Cy5-labeled) or control aptamers (FITC-labeled) and subjected to flow cytometry analysis (n = 5). (B) EPCs or 293FT cells were separately incubated with control aptamers (Ctrl AT, FITC-labeled) or CD31 aptamers (AT-1, Cy5-labeled, 200 nM) with or without 0.2 mM dextran sulfate and subjected to flow cytometry. The overlap of histograms from 0 and 0.2 mM dextran sulfate experiments is shown (n = 5). (C) The mixture of EPCs and 293FT cells was incubated with CD31 aptamers (AT-1, Cy5-labeled, 200 nM) and various concentrations (0, 0.2, 1, and 5 mM) of dextran sulfate, followed by flow cytometry analysis (n = 3). (D) EPCs were incubated with CD31 antibodies (FITC-labeled) alone, CD31 aptamers alone (AT-1, Cy5-labeled, 200 nM), or with both CD31 antibodies and CD31 aptamers and subjected to flow cytometry analysis. Two-dimensional plots are shown (n = 5).
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pone.0131785.g002: Dextran sulfate effectively reduces the non-specific interaction of CD31 aptamers.(A) 293FT cells were incubated with various concentrations (0, 0.2, 2, 20, and 200 nM) of CD31 aptamer clone 1 (AT-1, Cy5-labeled) or control aptamers (FITC-labeled) and subjected to flow cytometry analysis (n = 5). (B) EPCs or 293FT cells were separately incubated with control aptamers (Ctrl AT, FITC-labeled) or CD31 aptamers (AT-1, Cy5-labeled, 200 nM) with or without 0.2 mM dextran sulfate and subjected to flow cytometry. The overlap of histograms from 0 and 0.2 mM dextran sulfate experiments is shown (n = 5). (C) The mixture of EPCs and 293FT cells was incubated with CD31 aptamers (AT-1, Cy5-labeled, 200 nM) and various concentrations (0, 0.2, 1, and 5 mM) of dextran sulfate, followed by flow cytometry analysis (n = 3). (D) EPCs were incubated with CD31 antibodies (FITC-labeled) alone, CD31 aptamers alone (AT-1, Cy5-labeled, 200 nM), or with both CD31 antibodies and CD31 aptamers and subjected to flow cytometry analysis. Two-dimensional plots are shown (n = 5).

Mentions: Although CD31 aptamers showed strong interaction with EPCs, the specificity of the interaction was not confirmed. In flow cytometry analysis with CD31 antibodies, 293FT cells did not show expression of CD31 (S1 Fig). When 293FT cells were incubated with CD31 aptamer clone 1 at various concentrations from 0.2 to 200 nM, CD31 aptamers showed non-specific interaction with 293FT cells whereas control aptamers did not show interaction (Fig 2A). To resolve the non-specific interaction of CD31 aptamers, we introduced dextran sulfate, which is used for controlling hybridization temperature and blocking non-specific interaction in polymerase chain reaction, as an anionic competitor to incubation of CD31 aptamers with EPCs or 293FT cells [31, 32]. Without dextran sulfate, CD31 aptamers showed interaction with both EPCs and 293FT cells. Addition of 0.2 mM dextran sulfate did not inhibit the specific interaction of CD31 aptamers with EPCs. However, the interaction of CD31 aptamers with 293FT cells was completely abolished to the level of the control aptamers (Fig 2B). Incubation of CD31 aptamers with the mixture of EPCs and 293FT cells without dextran sulfate did not distinguish two populations (Fig 2C). However, addition of 0.2 mM dextran sulfate to the mixture of EPCs and 293FT cells clearly separated two peaks, indicating resolution of the non-specific interaction. Higher concentrations of dextran sulfate, such as 1 and 5 mM, inhibited the specific interaction of CD31 aptamers, suggesting that 0.2 mM dextran sulfate is the optimal concentration for blocking the non-specific interaction but allowing the specific interaction. The same patterns were observed with different clones of CD31 aptamers (S2 Fig). Single- or double-staining of EPCs with CD31 antibodies and/or CD31 aptamers in the presence of 0.2 mM dextran sulfate showed that CD31 aptamers are as efficient as CD31 antibodies in detecting EPCs (Fig 2D). These results demonstrate that 0.2 mM dextran sulfate resolves the non-specific interaction of CD31 aptamers and is required for application of CD31 aptamers in recognition of EPCs.


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)

Dextran sulfate effectively reduces the non-specific interaction of CD31 aptamers.(A) 293FT cells were incubated with various concentrations (0, 0.2, 2, 20, and 200 nM) of CD31 aptamer clone 1 (AT-1, Cy5-labeled) or control aptamers (FITC-labeled) and subjected to flow cytometry analysis (n = 5). (B) EPCs or 293FT cells were separately incubated with control aptamers (Ctrl AT, FITC-labeled) or CD31 aptamers (AT-1, Cy5-labeled, 200 nM) with or without 0.2 mM dextran sulfate and subjected to flow cytometry. The overlap of histograms from 0 and 0.2 mM dextran sulfate experiments is shown (n = 5). (C) The mixture of EPCs and 293FT cells was incubated with CD31 aptamers (AT-1, Cy5-labeled, 200 nM) and various concentrations (0, 0.2, 1, and 5 mM) of dextran sulfate, followed by flow cytometry analysis (n = 3). (D) EPCs were incubated with CD31 antibodies (FITC-labeled) alone, CD31 aptamers alone (AT-1, Cy5-labeled, 200 nM), or with both CD31 antibodies and CD31 aptamers and subjected to flow cytometry analysis. Two-dimensional plots are shown (n = 5).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131785.g002: Dextran sulfate effectively reduces the non-specific interaction of CD31 aptamers.(A) 293FT cells were incubated with various concentrations (0, 0.2, 2, 20, and 200 nM) of CD31 aptamer clone 1 (AT-1, Cy5-labeled) or control aptamers (FITC-labeled) and subjected to flow cytometry analysis (n = 5). (B) EPCs or 293FT cells were separately incubated with control aptamers (Ctrl AT, FITC-labeled) or CD31 aptamers (AT-1, Cy5-labeled, 200 nM) with or without 0.2 mM dextran sulfate and subjected to flow cytometry. The overlap of histograms from 0 and 0.2 mM dextran sulfate experiments is shown (n = 5). (C) The mixture of EPCs and 293FT cells was incubated with CD31 aptamers (AT-1, Cy5-labeled, 200 nM) and various concentrations (0, 0.2, 1, and 5 mM) of dextran sulfate, followed by flow cytometry analysis (n = 3). (D) EPCs were incubated with CD31 antibodies (FITC-labeled) alone, CD31 aptamers alone (AT-1, Cy5-labeled, 200 nM), or with both CD31 antibodies and CD31 aptamers and subjected to flow cytometry analysis. Two-dimensional plots are shown (n = 5).
Mentions: Although CD31 aptamers showed strong interaction with EPCs, the specificity of the interaction was not confirmed. In flow cytometry analysis with CD31 antibodies, 293FT cells did not show expression of CD31 (S1 Fig). When 293FT cells were incubated with CD31 aptamer clone 1 at various concentrations from 0.2 to 200 nM, CD31 aptamers showed non-specific interaction with 293FT cells whereas control aptamers did not show interaction (Fig 2A). To resolve the non-specific interaction of CD31 aptamers, we introduced dextran sulfate, which is used for controlling hybridization temperature and blocking non-specific interaction in polymerase chain reaction, as an anionic competitor to incubation of CD31 aptamers with EPCs or 293FT cells [31, 32]. Without dextran sulfate, CD31 aptamers showed interaction with both EPCs and 293FT cells. Addition of 0.2 mM dextran sulfate did not inhibit the specific interaction of CD31 aptamers with EPCs. However, the interaction of CD31 aptamers with 293FT cells was completely abolished to the level of the control aptamers (Fig 2B). Incubation of CD31 aptamers with the mixture of EPCs and 293FT cells without dextran sulfate did not distinguish two populations (Fig 2C). However, addition of 0.2 mM dextran sulfate to the mixture of EPCs and 293FT cells clearly separated two peaks, indicating resolution of the non-specific interaction. Higher concentrations of dextran sulfate, such as 1 and 5 mM, inhibited the specific interaction of CD31 aptamers, suggesting that 0.2 mM dextran sulfate is the optimal concentration for blocking the non-specific interaction but allowing the specific interaction. The same patterns were observed with different clones of CD31 aptamers (S2 Fig). Single- or double-staining of EPCs with CD31 antibodies and/or CD31 aptamers in the presence of 0.2 mM dextran sulfate showed that CD31 aptamers are as efficient as CD31 antibodies in detecting EPCs (Fig 2D). These results demonstrate that 0.2 mM dextran sulfate resolves the non-specific interaction of CD31 aptamers and is required for application of CD31 aptamers in recognition of EPCs.

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