The proper criteria for identification and sorting of very small embryonic-like stem cells, and some nomenclature issues

Abstract

Evidence has accumulated that both murine and human adult tissues contain early-development stem cells with a broader differentiation potential than other adult monopotent stem cells. These cells, being pluripotent or multipotent, exist at different levels of specification and most likely represent overlapping populations of cells that, depending on the isolation strategy, ex vivo expansion protocol, and markers employed for their identification, have been given different names. In this review, we will discuss a population of very small embryonic-like stem cells (VSELs) in the context of other stem cells that express pluripotent/multipotent markers isolated from adult tissues as well as review the most current, validated working criteria on how to properly identify and isolate these very rare cells. VSELs have been successfully purified in several laboratories; however, a few have failed to isolate them, which has raised some unnecessary controversy in the field. Therefore, in this short review, we will address the most important reasons that some investigators have experienced problems in isolating these very rare cells and discuss some still unresolved challenges which should be overcome before these cells can be widely employed in the clinic.

FIG. 1.

Comparison of a correct and incorrect gating strategy for murine BM very small embryonic-like stem cells (VSELs). (A) The correct gating strategy to analyze or sort murine BM-derived VSELs by FACS. Cells were fixed and stained with 7AAD to show nucleated events in gate P1. Gate P2 includes small, agranular cells. Gate P3 includes Sca-1⁺ Lin⁻ cells, which are visualized on the next dot plot as CD45-negative (VSELs) and CD45-positive cells (HSCs). Expanding this gate into lineage^dim population will result in contamination of VSELs by erythroblasts. Percentage shows the average content of each cellular subpopulation among total BM nucleated cells after fixation procedure and staining with 7ADD to gate for nucleated cells only. (B) An incorrect gating strategy (we followed the sorting procedure employed by Szade et al. [32]). As shown, this group employed extended regions P2, P3, and P4, (indicated by arrows) which resulted in enrichment of erythroblasts. (C) Expression of erythroblastic markers: CD71 and Ter-119 (markers of early-stage and more differentiated erythroblasts) in cells sorted by employing correct (A) and incorrect (B) sorting strategies. Cells enclosed by circles in the lower dot plot indicate an erythroblast population that contaminates the VSELs, which is not observed in cells sorted by the correct strategy (upper dot plot).

FIG. 2.

FACS analysis of umbilical cord blood (UCB) CD133⁺Lin⁻CD45⁻ VSELs. UCB VSELs are very rare cells, and their content varies, sometimes significantly, between UCB units. Upper panel–P1–shows DNA positive events shown in P2 as SSC versus FSC dot plot. P3–includes Lin⁻ CD45⁻ cells. These human UCB Lin⁻CD45⁻ cells were subsequently evaluated by FACS for the expression of CD133 antigen. The lower left panel illustrates an analysis for the presence of CD133⁺ cells in cells from P1/P2/P3 by employing a histogram, and the right panel is a visualization of these rare cells by dot plot. Danova-Alt et al. [31], in their recent studies, concluded that CD133⁺CD45⁻Lin⁻ cells as well as CD34⁺CD45⁻Lin⁻ cells do not exist in UCB. In fact, one of the reasons that these rare stem cell populations were overlooked by Danova-Alt and colleagues is they employed histograms instead of dot plot cytograms [31].

FIG. 3.

Hypothetical relation of VSELs to other multi/pluripotent stem cells identified in adult bone marrow (BM), peripheral blood (PB), and UCB. (A) VSELs and other multi/pluripotent stem cells identified in hematopoietic tissues are independent populations of stem cells. (B) VSELs are the most primitive small dormant stem cells that on proper activation give rise to expanding other larger multi/pluripotent stem cells. (C) VSELs are the most primitive small dormant stem cells that on proper activation give rise to other larger multi/pluripotent overlapping stem cell populations. We hypothesize that it is the most likely scenario.