Morphological characterization of very small embryonic-like stem cells (VSELs) by ImageStream system analysis.
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.
Zuba-Surma
Ewa K
EK
Stem Cell Biology Institute, University of Louisville, Louisville, KY 40202, USA. mzrata01@louisville.edu
Kucia
Magdalena
M
Abdel-Latif
Ahmed
A
Dawn
Buddhadeb
B
Hall
Brian
B
Singh
Rajesh
R
Lillard
James W
JW
Ratajczak
Mariusz Z
MZ
D000818:Animals; D001853:Bone Marrow / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D048429:Cell Size; D003593:Cytoplasm / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000648:ultrastructure*; D005434:Flow Cytometry; D006098:Granulocytes / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000648:ultrastructure; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal; D050814:Octamer Transcription Factor-3 / Q000378:metabolism
D050814:Octamer Transcription Factor-3