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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 interact with human EPCs.(A) Flow cytometry analysis of EPCs after individual incubation with various concentrations (0, 0.2, 2, 20, and 200 nM) of three CD31 aptamer clones (AT-1, AT-2, and AT-3, Cy5-labeled) is shown. (B) Flow cytometry analysis of EPCs after incubation with various concentrations (0, 0.2, 2, 20, and 200 nM) of control aptamers (FITC-labeled) is shown (n = 5).
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pone.0131785.g001: CD31 aptamers interact with human EPCs.(A) Flow cytometry analysis of EPCs after individual incubation with various concentrations (0, 0.2, 2, 20, and 200 nM) of three CD31 aptamer clones (AT-1, AT-2, and AT-3, Cy5-labeled) is shown. (B) Flow cytometry analysis of EPCs after incubation with various concentrations (0, 0.2, 2, 20, and 200 nM) of control aptamers (FITC-labeled) is shown (n = 5).

Mentions: In targeting distinct cells with functional cargo in complex mixtures of cells, aptamers can provide advantages over antibodies, which are most widely used in scientific and clinical applications, in the short generation time due to the entire chemical process and the easiness of modification with functional groups [29]. Aptamers are generated through an in vitro process termed SELEX, and a conservative estimate of the success rate is ~50% [30]. In addition, application of in vitro selected aptamers to cells requires fine calibration. Among CD31 aptamer clones selected using the purified extracellular domain of CD31, we tested three clones that showed the highest affinity in the application of recognizing CD31 on the cell surface. EPCs showed high expression of CD31 in analysis by flow cytometry using CD31 antibodies (S1 Fig). In application of CD31 aptamers to EPCs at various concentrations (0.2, 2, 20, and 200 nM), clones 1, 2, and 3 (AT-1, AT-2, and AT-3), each of which was labeled with Cy5, showed interaction with EPCs (Fig 1A). The intensity of the signal from CD31 aptamers continued to increase from 0.2 to 200 nM, and 400 nM treatment did not further increase the signal intensity (Fig 1A, data not shown). Among them, clone 1 (AT-1) showed the strongest signal, and we proceeded with the rest of the experiments using CD31 aptamer clone 1, if not stated otherwise (S1 Table). Incubation of ECPs with control scrambled EGFR-FITC aptamers did not generate a positive signal (Fig 1B). These results suggest that we generated aptamers that recognize CD31 on the surface 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)

CD31 aptamers interact with human EPCs.(A) Flow cytometry analysis of EPCs after individual incubation with various concentrations (0, 0.2, 2, 20, and 200 nM) of three CD31 aptamer clones (AT-1, AT-2, and AT-3, Cy5-labeled) is shown. (B) Flow cytometry analysis of EPCs after incubation with various concentrations (0, 0.2, 2, 20, and 200 nM) of control aptamers (FITC-labeled) is shown (n = 5).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131785.g001: CD31 aptamers interact with human EPCs.(A) Flow cytometry analysis of EPCs after individual incubation with various concentrations (0, 0.2, 2, 20, and 200 nM) of three CD31 aptamer clones (AT-1, AT-2, and AT-3, Cy5-labeled) is shown. (B) Flow cytometry analysis of EPCs after incubation with various concentrations (0, 0.2, 2, 20, and 200 nM) of control aptamers (FITC-labeled) is shown (n = 5).
Mentions: In targeting distinct cells with functional cargo in complex mixtures of cells, aptamers can provide advantages over antibodies, which are most widely used in scientific and clinical applications, in the short generation time due to the entire chemical process and the easiness of modification with functional groups [29]. Aptamers are generated through an in vitro process termed SELEX, and a conservative estimate of the success rate is ~50% [30]. In addition, application of in vitro selected aptamers to cells requires fine calibration. Among CD31 aptamer clones selected using the purified extracellular domain of CD31, we tested three clones that showed the highest affinity in the application of recognizing CD31 on the cell surface. EPCs showed high expression of CD31 in analysis by flow cytometry using CD31 antibodies (S1 Fig). In application of CD31 aptamers to EPCs at various concentrations (0.2, 2, 20, and 200 nM), clones 1, 2, and 3 (AT-1, AT-2, and AT-3), each of which was labeled with Cy5, showed interaction with EPCs (Fig 1A). The intensity of the signal from CD31 aptamers continued to increase from 0.2 to 200 nM, and 400 nM treatment did not further increase the signal intensity (Fig 1A, data not shown). Among them, clone 1 (AT-1) showed the strongest signal, and we proceeded with the rest of the experiments using CD31 aptamer clone 1, if not stated otherwise (S1 Table). Incubation of ECPs with control scrambled EGFR-FITC aptamers did not generate a positive signal (Fig 1B). These results suggest that we generated aptamers that recognize CD31 on the surface 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