title: 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(-)FSC(low) 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(-)FSC(low) 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(-)FSC(low) 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(-)FSC(low) 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.
authors.affiliation: Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Szade
authors.forename: Karolina
authors.initials: K
authors.lastname: Bukowska-Strakova
authors.forename: Witold Norbert
authors.initials: WN
authors.lastname: Nowak
authors.forename: Agata
authors.initials: A
authors.lastname: Szade
authors.forename: Neli
authors.initials: N
authors.lastname: Kachamakova-Trojanowska
authors.forename: Monika
authors.initials: M
authors.lastname: Zukowska
authors.forename: Alicja
authors.initials: A
authors.lastname: Jozkowicz
authors.forename: Jozef
authors.initials: J
authors.lastname: Dulak
mesh_terms: D000818:Animals; D000950:Antigens, Ly / Q000378:metabolism; D001854:Bone Marrow Cells / Q000378:metabolism*; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D018920:Coculture Techniques; D005434:Flow Cytometry; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype
chemical_list: D000950:Antigens, Ly; C074650:Ly6a protein, mouse; D008565:Membrane Proteins; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D017493:Leukocyte Common Antigens; C516770:Ptprc protein, mouse