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

Zuba-Surma EK, Kucia M, Abdel-Latif A, Dawn B, Hall B, Singh R, Lillard JW, Ratajczak MZ - J. Cell. Mol. Med. (2007)

Bottom Line: 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 microm in diameter).They also exhibit higher nuclear to cytoplasmic ratio and lower cytoplasmic area as compared with HSCs and mature granulocytes.Thus, FACS-based sorting strategies should consider that adult tissues harbour small primitive cells that are larger than platelets and smaller than erythrocytes.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Institute, University of Louisville, Louisville, KY 40202, USA. mzrata01@louisville.edu

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 microm 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.

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Analysis of nuclear to cytoplasmic ratio by ImageStream system. Single, round cells from region R1 (A) were visualized based on their nuclear to cytoplasm ratio and Lin markers expression (X- and Y- axis, respectively (B). Cellular populations were gated including Lin+ cells with low nuclear to cytoplasmic ratio (0.936 ± 0.016) (region R3, red) and Lin− cells with high N/C ratio (3.485 ± 0.248) (region R2, orange). Objects from region R2 were farther analysed for their CD45 and Sca-1 expression (X- and Y-axis, respectively (B). Cells with VSELs' phenotype (Sca-1+/Lin−/CD45−) and characterized by higher N/C ratio (1.471(0.171) were included in region R4 (magenta; C) and visualized on the other plots as diamonds (magenta). N/C ratio was calculated as nuclear area divided by cytoplasmic area computed from nuclear (7-AAD) and brightfield images. Signals of brightfield, Lin-PE and 7-AAD were collected by the IS in channels 2, 4 and 5, respectively. Mean (± S.E.M.) values of N/C ratio were calculated using IDEAS software.
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fig06: Analysis of nuclear to cytoplasmic ratio by ImageStream system. Single, round cells from region R1 (A) were visualized based on their nuclear to cytoplasm ratio and Lin markers expression (X- and Y- axis, respectively (B). Cellular populations were gated including Lin+ cells with low nuclear to cytoplasmic ratio (0.936 ± 0.016) (region R3, red) and Lin− cells with high N/C ratio (3.485 ± 0.248) (region R2, orange). Objects from region R2 were farther analysed for their CD45 and Sca-1 expression (X- and Y-axis, respectively (B). Cells with VSELs' phenotype (Sca-1+/Lin−/CD45−) and characterized by higher N/C ratio (1.471(0.171) were included in region R4 (magenta; C) and visualized on the other plots as diamonds (magenta). N/C ratio was calculated as nuclear area divided by cytoplasmic area computed from nuclear (7-AAD) and brightfield images. Signals of brightfield, Lin-PE and 7-AAD were collected by the IS in channels 2, 4 and 5, respectively. Mean (± S.E.M.) values of N/C ratio were calculated using IDEAS software.

Mentions: During BM-derived VSEL analysis, shown in Figures 4 and 6, the parameters of acquisition were attuned to detect and analyse very small events containing nuclei, which were negative for Linage markers and CD45 (i.e. Lin−/CD45− cells). For this purpose the settings and threshold of the instrument were adjusted to exclude most of the Lin+/CD45+ events by decreasing the peak upper limit for channels detecting fluorescence signals from CD45 and Lin (FITC- channel 3 and PE- channel 4, respectively). Simultaneously, IS was configured to detect even very small events containing DNA, defined in the protocol as small areas of brightfield containing a nucleus. Because of these settings, not all BM-derived cells were detected by IS and the majority CD45+ and Lin+ events were not analysed. VSELs were detected among the Sca-1+/Lin−/CD45−objects (Figs. 4 and 6).


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

Zuba-Surma EK, Kucia M, Abdel-Latif A, Dawn B, Hall B, Singh R, Lillard JW, Ratajczak MZ - J. Cell. Mol. Med. (2007)

Analysis of nuclear to cytoplasmic ratio by ImageStream system. Single, round cells from region R1 (A) were visualized based on their nuclear to cytoplasm ratio and Lin markers expression (X- and Y- axis, respectively (B). Cellular populations were gated including Lin+ cells with low nuclear to cytoplasmic ratio (0.936 ± 0.016) (region R3, red) and Lin− cells with high N/C ratio (3.485 ± 0.248) (region R2, orange). Objects from region R2 were farther analysed for their CD45 and Sca-1 expression (X- and Y-axis, respectively (B). Cells with VSELs' phenotype (Sca-1+/Lin−/CD45−) and characterized by higher N/C ratio (1.471(0.171) were included in region R4 (magenta; C) and visualized on the other plots as diamonds (magenta). N/C ratio was calculated as nuclear area divided by cytoplasmic area computed from nuclear (7-AAD) and brightfield images. Signals of brightfield, Lin-PE and 7-AAD were collected by the IS in channels 2, 4 and 5, respectively. Mean (± S.E.M.) values of N/C ratio were calculated using IDEAS software.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3823490&req=5

fig06: Analysis of nuclear to cytoplasmic ratio by ImageStream system. Single, round cells from region R1 (A) were visualized based on their nuclear to cytoplasm ratio and Lin markers expression (X- and Y- axis, respectively (B). Cellular populations were gated including Lin+ cells with low nuclear to cytoplasmic ratio (0.936 ± 0.016) (region R3, red) and Lin− cells with high N/C ratio (3.485 ± 0.248) (region R2, orange). Objects from region R2 were farther analysed for their CD45 and Sca-1 expression (X- and Y-axis, respectively (B). Cells with VSELs' phenotype (Sca-1+/Lin−/CD45−) and characterized by higher N/C ratio (1.471(0.171) were included in region R4 (magenta; C) and visualized on the other plots as diamonds (magenta). N/C ratio was calculated as nuclear area divided by cytoplasmic area computed from nuclear (7-AAD) and brightfield images. Signals of brightfield, Lin-PE and 7-AAD were collected by the IS in channels 2, 4 and 5, respectively. Mean (± S.E.M.) values of N/C ratio were calculated using IDEAS software.
Mentions: During BM-derived VSEL analysis, shown in Figures 4 and 6, the parameters of acquisition were attuned to detect and analyse very small events containing nuclei, which were negative for Linage markers and CD45 (i.e. Lin−/CD45− cells). For this purpose the settings and threshold of the instrument were adjusted to exclude most of the Lin+/CD45+ events by decreasing the peak upper limit for channels detecting fluorescence signals from CD45 and Lin (FITC- channel 3 and PE- channel 4, respectively). Simultaneously, IS was configured to detect even very small events containing DNA, defined in the protocol as small areas of brightfield containing a nucleus. Because of these settings, not all BM-derived cells were detected by IS and the majority CD45+ and Lin+ events were not analysed. VSELs were detected among the Sca-1+/Lin−/CD45−objects (Figs. 4 and 6).

Bottom Line: 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 microm in diameter).They also exhibit higher nuclear to cytoplasmic ratio and lower cytoplasmic area as compared with HSCs and mature granulocytes.Thus, FACS-based sorting strategies should consider that adult tissues harbour small primitive cells that are larger than platelets and smaller than erythrocytes.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell Biology Institute, University of Louisville, Louisville, KY 40202, USA. mzrata01@louisville.edu

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 microm 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.

Show MeSH
Related in: MedlinePlus