Very small embryonic-like stem cells in cardiovascular repair

Abstract

Adult bone marrow (BM) harbors several small populations of cells which may contribute to cardiac and endothelial repair, such as endothelial progenitor cells (EPCs), mesenchymal stromal cells (MSCs) and very small embryonic-like cells (VSELs) expressing several markers of pluripotent stem cells (PSCs), such as Oct-4, Nanog and SSEA-1. Such cells were identified in mice bone marrow, peripheral blood and solid organs as well as in umbilical cord blood (UCB) and peripheral blood (PB) in humans. The adult BM-derived VSELs may undergo differentiation into cells derived for all three germ layers, including cardiomyocytes and vascular endothelial cells. VSELs can be isolated using a multiparameter live cell sorting technique with special gating strategy based on their small size, expression of stem cell markers (Sca-1 in mice, CXCR4 and CD133 in humans) and absence of hematopoietic lineage markers (CD45(-) Lin(-)). Experiments in murine models of myocardial infarction (MI) demonstrated population of VSELs expressed also early markers of cardiac and endothelial lineages (GATA-4, Nkx2.5/Csx, VE-cadherin, von Willebrand factor) which migrated to stromal-derived factor-1 (SDF-1) and other chemoattractant gradient and underwent rapid mobilization into peripheral blood in experimental MI mice models. Recently, we demonstrated the mobilization of VSELs expressing PSC, early cardiac and endothelial markers in patients with acute MI. In addition to BM, VSELs were also identified in several murine solid organs including the heart and brain, as well as in umbilical cord blood and peripheral blood in adult humans. We hypothesized that VSELs are quiescent progeny of epiblast-derived PSCs that are deposited during organogenesis in developing organs. In experimental MI intramyocardial injection of VSELs was more efficient than that of HSCs at improving left ventricular ejection fraction and attenuation of myocardial hypertrophy. VSELs can be useful in translational studies of cardiovascular repair.

Fig. 1. Gating strategy for isolating VSELs by fluorescence- activated cell sorting (FACS)

Human VSELs were isolated from umbilical cord blood (CB)-derived total nucleated cells (TNCs) stained for: i) CD45 panleukocytic antigen, ii) hematopoietic lineages markers (Lineage/ Lin) and iii) CD133 stem cell antigen. Based on the size predefined beads (standard diameters of 1, 2, 4, 6, 10, and 15 µm) the gate R1 was set up to include all objects larger than 2µm (Panel A). TNCs were further visualized on the dot plot including region R1 and showing forward scatter (FSC) vs. side scatter (SSC) signals of cells that are related to the size and granularity/complexity of the cell, respectively (Panel B). Cells from region R1 were analyzed for hematopoietic lineages markers expression and only Lin⁻ events were included into region R2. Population from region R2 was subsequently visualized based on CD45 and CD133 antigens (Panel D). Lin⁻/CD45⁺/CD133⁺ cells (VSELs) were sorted as events enclosed in logical gate including regions R1, R2 and R4, while Lin⁻/CD45⁻/CD133⁺ hematopoietic stem/progenitor cells (HSPCs) from gate including regions R1, R2 and R3. Percentages show the average content of each cellular subpopulation (± SEM) in total nucleated cells.

Fig. 2. Representative images of human peripheral blood- derived VSEL and HSPC by ImageStreamX system

Human blood cells were stained for markers distinguishing VSELs such as: i) CD45 panleukocytic antigen (APC-Cy7, cyan), ii) hematopoietic lineages markers (FITC, green) and iii) stem cell antigens CD133 (PE, yellow) and CD34 (APC, violet). Nuclei were stained with Hoechst 33342 dye. Images were collected by imaging flow cytometer – ImageStreamX system. VSELs and HSPCs were distinguished based on CD45 antigen expression.

Fig. 3. The mechanism of mobilization and homing of VSELs in acute myocardial infarction and ischemic stroke

Ischemia induces increased expression of chemoattractants [phospholipids (such as sphingosine-1-phosphate), chemokines (SDF-1), growth factors (VEGF, HGF), cytokines (LIF), and activation of complement cascade]. In healthy subjects very low number of circulating VSELs can be detected in peripheral blood and expression of tissue specific markers is low. After mobilization in the setting of acute ischemia VSELs are mobilized and the mRNA levels for pluripotent markers, early cardiac and neural markers is significantly increased. Tissue ischemia leading to cleavage of SDF-1/CXCR4 in bone marrow and increased expression of chemoattractants in the ischemic organ creates the reversal of chemoattractant gradient leading to the release of VSELs from the bone marrow niches and their homing to injured organ.