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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

Preparation of foreign material-free EPCs from human cord blood MNC culture by magnetic cell sorting isolation with CD31 aptamer and decoupling processes.(A) Two-week culture of cord blood MNCs were subjected to magnetic bead isolation process with CD31 aptamers, followed by decoupling from CD31 aptamers with decoupling buffer. Bright field images (high and low magnification) of the MNCs and the EPCs decoupled from CD31 aptamers are shown. Scale bar = 100 μm. (B, C) Endothelial characteristics of the EPCs decoupled from CD31 aptamers were determined by staining with DiI-Ac-LDL (B) or lectin (Ulex europaeus agglutinin I) (C). Fluorescence images were taken by confocal microscope (Olympus FluoView FV1000). Scale bar = 40 μm. Representative data from three independent experiments are shown.
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pone.0131785.g005: Preparation of foreign material-free EPCs from human cord blood MNC culture by magnetic cell sorting isolation with CD31 aptamer and decoupling processes.(A) Two-week culture of cord blood MNCs were subjected to magnetic bead isolation process with CD31 aptamers, followed by decoupling from CD31 aptamers with decoupling buffer. Bright field images (high and low magnification) of the MNCs and the EPCs decoupled from CD31 aptamers are shown. Scale bar = 100 μm. (B, C) Endothelial characteristics of the EPCs decoupled from CD31 aptamers were determined by staining with DiI-Ac-LDL (B) or lectin (Ulex europaeus agglutinin I) (C). Fluorescence images were taken by confocal microscope (Olympus FluoView FV1000). Scale bar = 40 μm. Representative data from three independent experiments are shown.

Mentions: EPCs were established from the culture of MNCs from cord blood for more than three weeks [12]. In two-week culture of cord blood MNCs, heterogeneous morphology of cells (Fig 5A) and existence of CD31-negative cells along with CD31-positive cells were observed (data not shown). To expedite the establishment of EPCs in culture, we isolated CD31-positive cells from two-week culture of cord blood MNCs with biotin-labled CD31 aptamers and streptavidin magnetic beads and decoupled CD31 aptamers to generate foreign material-free EPCs as described in S5 Fig. CD31 aptamer-isolated and decoupled cells from cord blood MNC culture generated homogeneous cells in the subsequent culture (Fig 5A). Flow cytometry analysis showed CD31 aptamer-isolated and decoupled cells were positive for EPC surface markers such as CD31 (99.7%), KDR (43.3%), and VE-cadherin (92.8%) (S6 Fig). CD31 aptamer-isolated and decoupled cells in the culture showed endothelial characteristics such as uptake of Ac-LDL and staining by lectin (Fig 5B and 5C). These results demonstrate that foreign material-free EPCs can be isolated from heterogeneous mixture of cells using CD31 aptamers.


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)

Preparation of foreign material-free EPCs from human cord blood MNC culture by magnetic cell sorting isolation with CD31 aptamer and decoupling processes.(A) Two-week culture of cord blood MNCs were subjected to magnetic bead isolation process with CD31 aptamers, followed by decoupling from CD31 aptamers with decoupling buffer. Bright field images (high and low magnification) of the MNCs and the EPCs decoupled from CD31 aptamers are shown. Scale bar = 100 μm. (B, C) Endothelial characteristics of the EPCs decoupled from CD31 aptamers were determined by staining with DiI-Ac-LDL (B) or lectin (Ulex europaeus agglutinin I) (C). Fluorescence images were taken by confocal microscope (Olympus FluoView FV1000). Scale bar = 40 μm. Representative data from three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131785.g005: Preparation of foreign material-free EPCs from human cord blood MNC culture by magnetic cell sorting isolation with CD31 aptamer and decoupling processes.(A) Two-week culture of cord blood MNCs were subjected to magnetic bead isolation process with CD31 aptamers, followed by decoupling from CD31 aptamers with decoupling buffer. Bright field images (high and low magnification) of the MNCs and the EPCs decoupled from CD31 aptamers are shown. Scale bar = 100 μm. (B, C) Endothelial characteristics of the EPCs decoupled from CD31 aptamers were determined by staining with DiI-Ac-LDL (B) or lectin (Ulex europaeus agglutinin I) (C). Fluorescence images were taken by confocal microscope (Olympus FluoView FV1000). Scale bar = 40 μm. Representative data from three independent experiments are shown.
Mentions: EPCs were established from the culture of MNCs from cord blood for more than three weeks [12]. In two-week culture of cord blood MNCs, heterogeneous morphology of cells (Fig 5A) and existence of CD31-negative cells along with CD31-positive cells were observed (data not shown). To expedite the establishment of EPCs in culture, we isolated CD31-positive cells from two-week culture of cord blood MNCs with biotin-labled CD31 aptamers and streptavidin magnetic beads and decoupled CD31 aptamers to generate foreign material-free EPCs as described in S5 Fig. CD31 aptamer-isolated and decoupled cells from cord blood MNC culture generated homogeneous cells in the subsequent culture (Fig 5A). Flow cytometry analysis showed CD31 aptamer-isolated and decoupled cells were positive for EPC surface markers such as CD31 (99.7%), KDR (43.3%), and VE-cadherin (92.8%) (S6 Fig). CD31 aptamer-isolated and decoupled cells in the culture showed endothelial characteristics such as uptake of Ac-LDL and staining by lectin (Fig 5B and 5C). These results demonstrate that foreign material-free EPCs can be isolated from heterogeneous mixture of cells using CD31 aptamers.

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