Morphological characterization of very small embryonic‐like stem cells (VSELs) by ImageStream system analysis

Abstract

Recently, our group purified a rare population of primitive Sca1+/Lin−/CD45− cells from murine bone marrow by employing multiparameter cell sorting. Based on flow cytometric and gene expression analysis, these cells have been shown to express several markers of embryonic stem cells and were accordingly termed Very Small Embryonic-Like stem cells (VSELs). In order to better characterize VSELs, we focused on their morphological parameters (e.g. diameter, nuclear to cytoplasmic ratio, cytoplasmic area) as well as expression of Oct-4. To examine the morphological features of VSELs, we employed a multi-dimensional approach, including (i) traditional flow cytometry, (ii) a novel approach, which is ImageStream (IS) cytometry and (iii) confocal microscopy. We demonstrate by all of the sensitive and precise methods employed, that VSELs are a population of very small cells, which are significantly smaller than haematopoetic stem cells (HSC) (3.63 ± 0.09 versus 6.54 ±0.17 μm in diameter). They also exhibit higher nuclear to cytoplasmic ratio and lower cytoplasmic area as compared with HSCs and mature granulocytes. Besides confirming the size characteristics, confocal microscopic analysis also confirmed that VSELs express Oct-4, a marker of pluripotent embryonic stem cells. Morphological examination reveals that VSELs are unusually small eukaryotic cells that posses several characteristics of embryonic cells. Thus, FACS-based sorting strategies should consider that adult tissues harbour small primitive cells that are larger than platelets and smaller than erythrocytes.

Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity

“…-Confocal microscope and transmission electron microscope (TEM) analysis of VSELs-We have also confirmed the very small size of VSELs, when compared to hematopoietic stem cells by confocal (Zuba-Surma et al, 2008a) as well transmission electron microscopy (TEM) (Kucia et al, 2006a). Confocal microscopic analysis showed that sorted Sca-1 + /Lin − /CD45 + HSCs are relatively larger (>6μm) and stain negative for Oct-4 In contrast, VSELs are small nucleated cells (<6μm), do not express CD45 antigen on the surface, and express the ESC transcription factor Oct-4 in the nucleus (Zuba-Surma et al, 2008a).…”

“…We found that 95% of Sca-1 + /Lin − / CD45 − (VSELs) are located within the 2-6 μm size range, while 86% of Sca-1 + /Lin − / CD45 + (HSCs) are found in the 6-10 μm size range. Thus, by employing flow cytometry and the size marker beads, we have confirmed that the majority of Sca-1 + /Lin − /CD45 − cells isolated from adult BM is unusually small (<6 μm) (Zuba-Surma et al, 2008a). Thus, VSELs are larger than peripheral blood platelets and smaller than erythrocytes.…”

“…The high resolution of ISS imaging enables the identification of objects as small as 1 μm in diameter (Ortyn et al, 2006). Employing ISS analysis, we confirmed with greater precision that murine VSELs are ~3.6 μm in diameter, while Sca-1 + /Lin − /CD45 + HSCs are larger at ~6.5 μm in diameter (Zuba-Surma et al, 2008a). Finally, we investigated the N/C ratio as well as the cytoplasmic area of VSELs in comparison with HSCs.…”

“…The Sca-1 + /Lin − events shown in region R2 (Figure 2) consist of 0.30 ± 0.05% of total analyzed BM nucleated cells on average. Cells from region R2 are subsequently sorted according to the expression of CD45 antigen as Sca-1 + /Lin − /CD45 − (region R3) and Sca-1 + /Lin − /CD45 + (region R4) subpopulations (Figure 2) that contain VSELs and HSCs, respectively (Kucia et al, 2006a;Zuba-Surma et al, 2008a). We found that VSELs comprise ~0.03% while HSCs are ~0.35% of total BM nucleated cells (Figure 2).…”

“…Confocal microscopic analysis showed that sorted Sca-1 + /Lin − /CD45 + HSCs are relatively larger (>6μm) and stain negative for Oct-4 In contrast, VSELs are small nucleated cells (<6μm), do not express CD45 antigen on the surface, and express the ESC transcription factor Oct-4 in the nucleus (Zuba-Surma et al, 2008a). Direct TEM analysis revealed that these cells display several features typical for ESCs such as small size, a large nucleus surrounded by a narrow rim of cytoplasm, and open-type chromatin (euchromatin) (Kucia et al, 2006a).…”