Mobilization of bone marrow-derived Oct-4+ SSEA-4+ very small embryonic-like stem cells in patients with acute myocardial infarction

Abstract

Objectives: This study sought to assess of the mobilization of nonhematopoietic very small embryonic-like stem cells (VSELs) in acute myocardial infarction (MI).

Background: Acute MI induces mobilization of bone marrow stem cells. Recently, a rare population of VSELs, expressing markers of embryonic pluripotent stem cells (PSCs), was identified in adult murine bone marrow and human umbilical cord blood.

Methods: Thirty-one patients with acute MI and 30 healthy subjects were enrolled. Blood was sampled on admission, after 24 h, and 5 days later. Erythrocytes were lysed and lin(-)CD45(-) VSELs were isolated using a live cell sorting system (FACSAria, Beckton Dickinson, San Jose, California).

Results: In healthy subjects the median number of circulating VSELs was very low (median 0.8 [range 0 to 1.3]) cells/microl. In acute MI, VSELs were mobilized early (median 2.7 [range 0.2 to 3.9] cells/microl; p < 0.001) and remained elevated after 24 h and 5 days (median 4.7 [range 0.2 to 6.4] cells/microl; p < 0.003, and median 2.6 [range 0.3 to 3.6] cells/microl; p < 0.03, respectively). The mobilization of VSEL was significantly reduced in patients older than 50 years and with diabetes in comparison with younger and nondiabetic patients. Circulating VSELs were small (7 to 8 microm) and enriched in the messenger ribonucleic acid of PSC markers (Oct-4, Nanog), cardiac lineage (GATA-4, Nkx2.5/Csx, MEF2C), and endothelial (VE-cadherin) markers. The presence of PSC markers (Oct-4, SSEA-4) and the chemokine receptor CXCR4 in circulating VSELs was confirmed at the protein level by immunofluorescent staining and ImageStream system (Amnis Corporation, Seattle, Washington) analysis.

Conclusions: Acute MI induced mobilization of VSELs expressing pluripotent markers, early cardiac and endothelial markers, and chemokine receptor CXCR4.

Figure 1. Procedure of isolation of VSEL from peripheral blood

Isolation of VSEL from peripheral blood nucleated cells by hypotnic lysis of erythrocytes and sterile, live cell sorting using FACSAria cell sorting system (Panel A). Sorting was based on the immunophenotype (lin⁻CXCR4⁺CD34⁺CD133⁺CD45⁻) of VSEL. Gating strategy enabled to include small events consistent with the size range of VSEL (5-7 μm). Events contained by gate P1 were characterized by the absence of lineage markers and presence of CD34, CD133 and CXCR4 (gate P2) and absence of CD45 (gate P3) which represent the population of VSEL (Panel B). Panel A. Procedure flowchart Panel B. Sorting of subpopulation of non-hematopoietic CD45⁻ lin⁻CD34⁺, lin⁻CD45⁻ CD133⁺ and lin⁻CD45⁻CXCR4⁺ VSEL.

Figure 1. Procedure of isolation of VSEL from peripheral blood

Isolation of VSEL from peripheral blood nucleated cells by hypotnic lysis of erythrocytes and sterile, live cell sorting using FACSAria cell sorting system (Panel A). Sorting was based on the immunophenotype (lin⁻CXCR4⁺CD34⁺CD133⁺CD45⁻) of VSEL. Gating strategy enabled to include small events consistent with the size range of VSEL (5-7 μm). Events contained by gate P1 were characterized by the absence of lineage markers and presence of CD34, CD133 and CXCR4 (gate P2) and absence of CD45 (gate P3) which represent the population of VSEL (Panel B). Panel A. Procedure flowchart Panel B. Sorting of subpopulation of non-hematopoietic CD45⁻ lin⁻CD34⁺, lin⁻CD45⁻ CD133⁺ and lin⁻CD45⁻CXCR4⁺ VSEL.

Figure 2. Mobilization of lin⁻CD133⁺CD45⁻ VSEL

Mobilization of cells shown as change in absolute number of cells per μL of peripheral blood in patients with acute MI in comparison to healthy controls.

Figure 3. Expression of pluripotent stem cells markers in circulating VSEL

Relative changes in expression of pluripotent stem cells markers in circulating VSEL isolated by sterile, live cell sorting from peripheral blood of patients with acute MI and healthy controls measured by real time RT-PCR. Expression of each gene showed as fold difference results compared to healthy subjects (n=15).

Figure 4. Analysis of VSELs by immunofluorescent staining and ImageStream System

Immunofluorescent staining of peripheral blood-derived lin⁻CD45⁻CD133⁺ VSELs sorted with FACSAria (Panel A). VSELs express SSEA-4, Oct-4 and CXCR4 at the protein level. CXCR4 and SSEA-4 are visualized in the cellular membrane and Oct-4 in the nucleus. Nuclei are stained with DAPI. Staining was performed on cells isolated from four independent sorts. (Panel B) Circulating human VSELs analyzed by ImageStream system. Expression of Oct-4 and SSEA-4 in lin⁻CD45⁻CXCR4⁺ VSELs was confirmed at the single cell level. The size of VSELs was compared to granulocytes and erythrocytes derived from peripheral blood of patients with acute MI. The size of the cells was calculated by IDEAS software. Scale bars indicate 10μm.

Figure 4. Analysis of VSELs by immunofluorescent staining and ImageStream System

Immunofluorescent staining of peripheral blood-derived lin⁻CD45⁻CD133⁺ VSELs sorted with FACSAria (Panel A). VSELs express SSEA-4, Oct-4 and CXCR4 at the protein level. CXCR4 and SSEA-4 are visualized in the cellular membrane and Oct-4 in the nucleus. Nuclei are stained with DAPI. Staining was performed on cells isolated from four independent sorts. (Panel B) Circulating human VSELs analyzed by ImageStream system. Expression of Oct-4 and SSEA-4 in lin⁻CD45⁻CXCR4⁺ VSELs was confirmed at the single cell level. The size of VSELs was compared to granulocytes and erythrocytes derived from peripheral blood of patients with acute MI. The size of the cells was calculated by IDEAS software. Scale bars indicate 10μm.

Figure 5. Expression of early cardiac and endothelial markers in circulating VSELs

Relative changes in expression of early cardiac and endothelial markers in circulating VSEL isolated by sterile, live cell sorting from peripheral blood of patients with acute MI and healthy controls measured by real time RT-PCR. Expression of each gene showed as fold difference results compared to healthy subjects (n=15).