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Cord blood Lin(-)CD45(-) embryonic-like stem cells are a heterogeneous population that lack self-renewal capacity.

ABSTRACT: Human umbilical cord blood (hUCB) has been proposed to contain not only haematopoietic stem cells, but also a rare pluripotent embryonic-like stem cell (ELSc) population that is negative for hematopoietic markers (Lin(-)CD45(-)) and expresses markers typical of pluripotent cells. The aim of this work was to isolate, characterise and expand this ELSc fraction from hUCB, as it may provide a valuable cell source for regenerative medicine applications. We found that we could indeed isolate a Lin(-)CD45(-) population of small cells (3-10 µm diameter) with a high nucleus to cytoplasm ratio that expressed the stem cell markers CD34 and CXCR4. However, in contrast to some previous reports, this fraction was not positive for CD133. Furthermore, although these cells expressed transcripts typical of pluripotent cells, such as SOX2, OCT3/4, and NANOG, they were not able to proliferate in any of the culture media known to support stem cell growth that we tested. Further analysis of the Lin(-)CD45(-) population by flow cytometry showed the presence of a Lin(-)CD45(-)Nestin(+) population that were also positive for CD34 (20%) but negative for CXCR4. These data suggest that the Lin(-)CD45(-) stem cell fraction present in the cord blood represents a small heterogeneous population with phenotypic characteristics of stem cells, including a Lin(-)CD45(-)Nestin(+) population not previously described. This study also suggests that heterogeneity within the Lin(-)CD45(-) cell fraction is the likely explanation for differences in the hUCB cell populations described by different groups that were isolated using different methods. These populations have been widely called "embryonic-like stem cell" on the basis of their phenotypical similarity to embryonic stem cells. However, the fact they do not seem to be able to self-renew casts some doubt on their identity, and warns against defining them as "embryonic-like stem cell" at this stage.

SUBMITTER: Alvarez-Gonzalez C

PROVIDER: S-EPMC3695943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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.

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Cord blood Lin(-)CD45(-) embryonic-like stem cells are a heterogeneous population that lack self-renewal capacity.

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