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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 isolate EPCs from the mixture of EPCs and 293FT cells and EPCs are decoupled from CD31 aptamers.(A) EPCs, 293FT cells, or the mixture of EPCs and 293FT cells were analyzed by flow cytometry after staining cells with FITC-labeled anti-human CD31 antibodies (n = 3). (B) The mixture of EPCs and 293FT cells was subjected to magnetic bead sorting using CD31 aptamers (AT-1, biotin-labeled) (upper panel) or CD31 antibodies (lower panel). (+) indicates the fraction with positive selection and (-) indicates the fraction with negative selection. Flow cytometry analysis of each fraction with CD31 antibodies (FITC-labeled) after isolation is shown (n = 3). (C) Bright field images of cultured cells in (+) fraction after magnetic bead isolation of the mixture of EPCs and 293FT cells with CD31 aptamers (upper panel) or CD31 antibodies (lower panel) are shown. Scale bar = 200 μm (n = 3). (D) Decoupling of EPCs from CD31 aptamer-EPC complexes by treatment with DxSO4. EPCs were decoupled from the CD31 aptamer-EPC complexes and recovery yield was calculated by comparing cell numbers before and after decoupling with DxSO4 (left panel) (n = 4). Flow cytometry analysis of decoupled cells with CD31 antibody is shown in the right panel. (E) Viability of EPCs before and after decoupling with DxSO4 is shown by flow cytometry analysis (n = 5).
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pone.0131785.g004: CD31 aptamers isolate EPCs from the mixture of EPCs and 293FT cells and EPCs are decoupled from CD31 aptamers.(A) EPCs, 293FT cells, or the mixture of EPCs and 293FT cells were analyzed by flow cytometry after staining cells with FITC-labeled anti-human CD31 antibodies (n = 3). (B) The mixture of EPCs and 293FT cells was subjected to magnetic bead sorting using CD31 aptamers (AT-1, biotin-labeled) (upper panel) or CD31 antibodies (lower panel). (+) indicates the fraction with positive selection and (-) indicates the fraction with negative selection. Flow cytometry analysis of each fraction with CD31 antibodies (FITC-labeled) after isolation is shown (n = 3). (C) Bright field images of cultured cells in (+) fraction after magnetic bead isolation of the mixture of EPCs and 293FT cells with CD31 aptamers (upper panel) or CD31 antibodies (lower panel) are shown. Scale bar = 200 μm (n = 3). (D) Decoupling of EPCs from CD31 aptamer-EPC complexes by treatment with DxSO4. EPCs were decoupled from the CD31 aptamer-EPC complexes and recovery yield was calculated by comparing cell numbers before and after decoupling with DxSO4 (left panel) (n = 4). Flow cytometry analysis of decoupled cells with CD31 antibody is shown in the right panel. (E) Viability of EPCs before and after decoupling with DxSO4 is shown by flow cytometry analysis (n = 5).

Mentions: EPCs have been shown to provide therapeutic benefits when transplanted into ischemic injury models [12, 34]. We tested whether CD31 aptamers could isolate EPCs from the mixture of cells. We prepared, the cell mixture comprised of ~72% EPCs and ~28% 293FT cells (Fig 4A). To test the efficiency of cell sorting with CD31 aptamers, the mixture of EPCs and 293FT cells was subjected to magnetic bead sorting using biotin-labeled CD31 aptamers and streptavidin magnetic beads. The mixture of EPCs and 293FT cells was also subjected to magnetic bead sorting using CD31 antibodies for comparison of the sorting efficiency. In analysis of post-sort fractions using CD31 antibodies, the EPC fraction isolated by CD31 antibodies showed 98.9% purity and 99.5% yield. The EPC fraction isolated by CD31 aptamers showed 97.6% purity and 94.2% yield, comparable to those of isolation by CD31 antibodies (Fig 4B). When isolated EPCs were subjected to culture, both EPC fractions from CD31 aptamer-isolation and CD31 antibody-isolation showed proliferating EPCs, whose morphology is distinct from that of 293FT cells (Fig 4C). These results demonstrate that CD31 aptamers can isolate EPCs with high purity and high yield comparable to those of CD31 antibodies from the mixture of cells.


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 isolate EPCs from the mixture of EPCs and 293FT cells and EPCs are decoupled from CD31 aptamers.(A) EPCs, 293FT cells, or the mixture of EPCs and 293FT cells were analyzed by flow cytometry after staining cells with FITC-labeled anti-human CD31 antibodies (n = 3). (B) The mixture of EPCs and 293FT cells was subjected to magnetic bead sorting using CD31 aptamers (AT-1, biotin-labeled) (upper panel) or CD31 antibodies (lower panel). (+) indicates the fraction with positive selection and (-) indicates the fraction with negative selection. Flow cytometry analysis of each fraction with CD31 antibodies (FITC-labeled) after isolation is shown (n = 3). (C) Bright field images of cultured cells in (+) fraction after magnetic bead isolation of the mixture of EPCs and 293FT cells with CD31 aptamers (upper panel) or CD31 antibodies (lower panel) are shown. Scale bar = 200 μm (n = 3). (D) Decoupling of EPCs from CD31 aptamer-EPC complexes by treatment with DxSO4. EPCs were decoupled from the CD31 aptamer-EPC complexes and recovery yield was calculated by comparing cell numbers before and after decoupling with DxSO4 (left panel) (n = 4). Flow cytometry analysis of decoupled cells with CD31 antibody is shown in the right panel. (E) Viability of EPCs before and after decoupling with DxSO4 is shown by flow cytometry analysis (n = 5).
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Related In: Results  -  Collection

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pone.0131785.g004: CD31 aptamers isolate EPCs from the mixture of EPCs and 293FT cells and EPCs are decoupled from CD31 aptamers.(A) EPCs, 293FT cells, or the mixture of EPCs and 293FT cells were analyzed by flow cytometry after staining cells with FITC-labeled anti-human CD31 antibodies (n = 3). (B) The mixture of EPCs and 293FT cells was subjected to magnetic bead sorting using CD31 aptamers (AT-1, biotin-labeled) (upper panel) or CD31 antibodies (lower panel). (+) indicates the fraction with positive selection and (-) indicates the fraction with negative selection. Flow cytometry analysis of each fraction with CD31 antibodies (FITC-labeled) after isolation is shown (n = 3). (C) Bright field images of cultured cells in (+) fraction after magnetic bead isolation of the mixture of EPCs and 293FT cells with CD31 aptamers (upper panel) or CD31 antibodies (lower panel) are shown. Scale bar = 200 μm (n = 3). (D) Decoupling of EPCs from CD31 aptamer-EPC complexes by treatment with DxSO4. EPCs were decoupled from the CD31 aptamer-EPC complexes and recovery yield was calculated by comparing cell numbers before and after decoupling with DxSO4 (left panel) (n = 4). Flow cytometry analysis of decoupled cells with CD31 antibody is shown in the right panel. (E) Viability of EPCs before and after decoupling with DxSO4 is shown by flow cytometry analysis (n = 5).
Mentions: EPCs have been shown to provide therapeutic benefits when transplanted into ischemic injury models [12, 34]. We tested whether CD31 aptamers could isolate EPCs from the mixture of cells. We prepared, the cell mixture comprised of ~72% EPCs and ~28% 293FT cells (Fig 4A). To test the efficiency of cell sorting with CD31 aptamers, the mixture of EPCs and 293FT cells was subjected to magnetic bead sorting using biotin-labeled CD31 aptamers and streptavidin magnetic beads. The mixture of EPCs and 293FT cells was also subjected to magnetic bead sorting using CD31 antibodies for comparison of the sorting efficiency. In analysis of post-sort fractions using CD31 antibodies, the EPC fraction isolated by CD31 antibodies showed 98.9% purity and 99.5% yield. The EPC fraction isolated by CD31 aptamers showed 97.6% purity and 94.2% yield, comparable to those of isolation by CD31 antibodies (Fig 4B). When isolated EPCs were subjected to culture, both EPC fractions from CD31 aptamer-isolation and CD31 antibody-isolation showed proliferating EPCs, whose morphology is distinct from that of 293FT cells (Fig 4C). These results demonstrate that CD31 aptamers can isolate EPCs with high purity and high yield comparable to those of CD31 antibodies from the mixture of cells.

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