Murine Bone Marrow Lin−Sca-1+CD45− Very Small Embryonic-Like (VSEL) Cells Are Heterogeneous Population Lacking Oct-4A Expression

Abstract

Murine very small embryonic-like (VSEL) cells, defined by the Lin−Sca-1+CD45− phenotype and small size, were described as pluripotent cells and proposed to be the most primitive hematopoietic precursors in adult bone marrow. Although their isolation and potential application rely entirely on flow cytometry, the immunophenotype of VSELs has not been extensively characterized. Our aim was to analyze the possible heterogeneity of Lin−Sca+CD45− population and investigate the extent to which VSELs characteristics may overlap with that of hematopoietic stem cells (HSCs) or endothelial progenitor cells (EPCs). The study evidenced that murine Lin−Sca-1+CD45− population was heterogeneous in terms of c-Kit and KDR expression. Accordingly, the c-Kit+KDR−, c-Kit−KDR+, and c-Kit−KDR− subpopulations could be distinguished, while c-Kit+KDR+ events were very rare. The c-Kit+KDR− subset contained almost solely small cells, meeting the size criterion of VSELs, in contrast to relatively bigger c-Kit−KDR+ cells. The c-Kit−KDR−FSClow subset was highly enriched in Annexin V-positive, apoptotic cells, hence omitted from further analysis. Importantly, using qRT-PCR, we evidenced lack of Oct-4A and Oct-4B mRNA expression either in whole adult murine bone marrow or in the sorted of Lin−Sca-1+CD45−FSClow population, even by single-cell qRT-PCR. We also found that the Lin−Sca-1+CD45−c-Kit+ subset did not exhibit hematopoietic potential in a single cell-derived colony in vitro assay, although it comprised the Sca-1+c-Kit+Lin− (SKL) CD34−CD45−CD105+ cells, expressing particular HSC markers. Co-culture of Lin−Sca-1+CD45−FSClow with OP9 cells did not induce hematopoietic potential. Further investigation revealed that SKL CD45−CD105+ subset consisted of early apoptotic cells with fragmented chromatin, and could be contaminated with nuclei expelled from erythroblasts. Concluding, murine bone marrow Lin−Sca-1+CD45−FSClow cells are heterogeneous population, which do not express the pluripotency marker Oct-4A. Despite expression of some hematopoietic markers by a Lin−Sca-1+CD45−c-Kit+KDR− subset of VSELs, they do not display hematopoietic potential in a clonogenic assay and are enriched in early apoptotic cells.

Human and Murine Very Small Embryonic-Like Cells Represent Multipotent Tissue Progenitors, In Vitro and In Vivo

“…Recently, several groups have reported that they have been unable to isolate VSEL cells and suggest that there are insufficient animal data to warrant human clinical trials [28][29][30]. There is no doubt that isolation of VSEL cells and their cultivation is a difficult endeavor.…”

Do Pluripotent Stem Cells Exist in Adult Mice as Very Small Embryonic Stem Cells?

“…Thus, their pluripotency should be reproducible in independent laboratories, a requirement for a scientific finding to be generally accepted (Jasny et al., 2011). However, recent studies have shown the lack of stem cell characteristics in VSELs isolated from mouse (Szade et al., 2013) or human cord blood (CB) (Danova-Alt et al., 2012), while another study confirmed the existence of mouse VSELs and their ability to give rise to lung cells (Kassmer et al., 2013). …”

Very Small Embryonic-Like Stem Cells: Are They Real?

“…Is one set of ideas going to triumph about the existence of VSELs or will there eventually be a resolution where both sides' hypotheses contain aspects that are correct? I actually don't think we know yet, but publications showing evidence for VSELs are cropping up in good journals [5][6][7][8][9]. That said, I do believe finding our way through this controversy has a ''going to be fun to watch'' outlook written all over it.…”