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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

Foreign material-free EPCs enhance restoration of blood flow and limb salvage from ischemic injury.(A) The foreign material-free EPCs were injected intramuscularly to injury site of nude mice with hindlimb ischemia (1×106 cells/injection). Representative images of mice with laser Doppler perfusion imaging (LDPI) on day 0 and day 28 after injection with HBSS buffer (n = 6) or EPCs (n = 6) are shown. (B) Time-course quantitative analysis of blood flow by LDPI of hindlimb ischemia-induced mice injected with EPCs or HBSS is shown. (C) Statistical analysis of necrosis score on day 28 is shown. (D) Immunostaining of CD31-positive capillaries (green) in salvaged hindlimbs on day 28 after intramuscular injection of HBSS or EPCs is shown. (E) Quantitative analysis of capillary density in salvaged limbs on day 28 by counting the number of CD31-positive capillaries per high-power field (HPF) is shown. * indicates p < 0.05 vs HBSS by Student’s t-test (n = 6 for EPC and n = 6 for HBSS respectively).
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pone.0131785.g006: Foreign material-free EPCs enhance restoration of blood flow and limb salvage from ischemic injury.(A) The foreign material-free EPCs were injected intramuscularly to injury site of nude mice with hindlimb ischemia (1×106 cells/injection). Representative images of mice with laser Doppler perfusion imaging (LDPI) on day 0 and day 28 after injection with HBSS buffer (n = 6) or EPCs (n = 6) are shown. (B) Time-course quantitative analysis of blood flow by LDPI of hindlimb ischemia-induced mice injected with EPCs or HBSS is shown. (C) Statistical analysis of necrosis score on day 28 is shown. (D) Immunostaining of CD31-positive capillaries (green) in salvaged hindlimbs on day 28 after intramuscular injection of HBSS or EPCs is shown. (E) Quantitative analysis of capillary density in salvaged limbs on day 28 by counting the number of CD31-positive capillaries per high-power field (HPF) is shown. * indicates p < 0.05 vs HBSS by Student’s t-test (n = 6 for EPC and n = 6 for HBSS respectively).

Mentions: CD31-postive cells isolated from peripheral blood or bone marrow were previously shown to contribute to the repair of ischemic vascular injuries [21, 22]. Therefore, we tested whether the foreign material-free CD31-positive cells decoupled from CD31 aptamer-conjugated microbeads could contribute to the vascular repair from ischemic injuries. Femoral artery of nude mice was ligated surgically to introduce ischemic injuries and the foreign material-free CD31-positive cells were injected intramuscularly to the site of ischemic injury on the following day (1 x 106 cells/injection). Mice were monitored up to day 28 after EPC injection with time-course measuring of blood flow. Analysis of mice on day 28 showed that EPC injection significantly improved blood flow and limb salvage compared with control HBSS injection (Fig 6A, 6B and 6C). Immunohistochemistry analysis of salvaged limbs showed that EPC injection increased CD31-positive capillaries compared with control HBSS injection (Fig 6D and 6E). These results demonstrate that EPCs established from heterogeneous culture of cord blood MNCs using CD31 aptamer isolation and decoupling protocol are functional and contribute to the recovery from ischemic injuries.


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)

Foreign material-free EPCs enhance restoration of blood flow and limb salvage from ischemic injury.(A) The foreign material-free EPCs were injected intramuscularly to injury site of nude mice with hindlimb ischemia (1×106 cells/injection). Representative images of mice with laser Doppler perfusion imaging (LDPI) on day 0 and day 28 after injection with HBSS buffer (n = 6) or EPCs (n = 6) are shown. (B) Time-course quantitative analysis of blood flow by LDPI of hindlimb ischemia-induced mice injected with EPCs or HBSS is shown. (C) Statistical analysis of necrosis score on day 28 is shown. (D) Immunostaining of CD31-positive capillaries (green) in salvaged hindlimbs on day 28 after intramuscular injection of HBSS or EPCs is shown. (E) Quantitative analysis of capillary density in salvaged limbs on day 28 by counting the number of CD31-positive capillaries per high-power field (HPF) is shown. * indicates p < 0.05 vs HBSS by Student’s t-test (n = 6 for EPC and n = 6 for HBSS respectively).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
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pone.0131785.g006: Foreign material-free EPCs enhance restoration of blood flow and limb salvage from ischemic injury.(A) The foreign material-free EPCs were injected intramuscularly to injury site of nude mice with hindlimb ischemia (1×106 cells/injection). Representative images of mice with laser Doppler perfusion imaging (LDPI) on day 0 and day 28 after injection with HBSS buffer (n = 6) or EPCs (n = 6) are shown. (B) Time-course quantitative analysis of blood flow by LDPI of hindlimb ischemia-induced mice injected with EPCs or HBSS is shown. (C) Statistical analysis of necrosis score on day 28 is shown. (D) Immunostaining of CD31-positive capillaries (green) in salvaged hindlimbs on day 28 after intramuscular injection of HBSS or EPCs is shown. (E) Quantitative analysis of capillary density in salvaged limbs on day 28 by counting the number of CD31-positive capillaries per high-power field (HPF) is shown. * indicates p < 0.05 vs HBSS by Student’s t-test (n = 6 for EPC and n = 6 for HBSS respectively).
Mentions: CD31-postive cells isolated from peripheral blood or bone marrow were previously shown to contribute to the repair of ischemic vascular injuries [21, 22]. Therefore, we tested whether the foreign material-free CD31-positive cells decoupled from CD31 aptamer-conjugated microbeads could contribute to the vascular repair from ischemic injuries. Femoral artery of nude mice was ligated surgically to introduce ischemic injuries and the foreign material-free CD31-positive cells were injected intramuscularly to the site of ischemic injury on the following day (1 x 106 cells/injection). Mice were monitored up to day 28 after EPC injection with time-course measuring of blood flow. Analysis of mice on day 28 showed that EPC injection significantly improved blood flow and limb salvage compared with control HBSS injection (Fig 6A, 6B and 6C). Immunohistochemistry analysis of salvaged limbs showed that EPC injection increased CD31-positive capillaries compared with control HBSS injection (Fig 6D and 6E). These results demonstrate that EPCs established from heterogeneous culture of cord blood MNCs using CD31 aptamer isolation and decoupling protocol are functional and contribute to the recovery from ischemic injuries.

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