CD133 Expression Strongly Correlates with the Phenotype of Very Small Embryonic-/Epiblast-Like Stem Cells

Abstract

CD133 antigen (prominin-1) is a useful cell surface marker of very small embryonic-like stem cells (VSELs). Antibodies against it, conjugated to paramagnetic beads or fluorochromes, are thus powerful biological tools for their isolation from human umbilical cord blood, mobilized peripheral blood, and bone marrow. VSELs are described with the following characteristics: (1) are slightly smaller than red blood cells; (2) display a distinct morphology, typified by a high nuclear/cytoplasmic ratio and an unorganized euchromatin; (3) become mobilized during stress situations into peripheral blood; (4) are enriched in the CD133(+)Lin(-)CD45(-) cell fraction in humans; and (5) express markers of pluripotent stem cells (e.g., Oct-4, Nanog, and stage-specific embryonic antigen-4). The most recent in vivo data from our and other laboratories demonstrated that human VSELs exhibit some characteristics of long-term repopulating hematopoietic stem cells and are at the top of the hierarchy in the mesenchymal lineage. However, still more labor is needed to characterize better at a molecular level these rare cells.

Fig. 9.1

Gating strategy for multiparameter sorting of human UCB-VSELs and UCB-HSCs by using CD133 antibody. UCB-derived cells were isolated by FACS by employing the following gating criteria. Panel 1: All events ≥2-μm are included in gate R1 after comparison with bead particles at standard diameters of 1, 2, 4, 6, 10, or 15 μm. Panel 2: UCB-derived total nucleated cells are visualized on a dot plot based on forward scatter (FS; x-axis) vs side scatter (SS; y-axis) signals. Panel 3: Cells from region R1 (panel 2) are further analyzed for Lin and CD133. Events for cells expressing Lin⁻ and CD133⁺ are included in region R3. Panel 4: The Lin⁻/CD133⁺ population from region R3 is subsequently analyzed based on CD45 antigen expression, and the CD45⁻ and CD45⁺ subpopulations were visualized with a dot plot showing CD133⁺Lin⁻CD45⁻ (VSELs, region R4) and CD133⁺Lin⁻CD45⁺ (HSCs, region R5) populations.

Fig. 9.2

Gating strategy for FACS analysis of UCB-VSELs and UCB-HSCs based on CD133 and CD45 expression and ALDH activity. UCB-derived CD133⁺ VSELs were isolated by FACS by employing the following gating criteria. Panel 1: UCB nucleated cell populations are stained using mAbs against human CD235a (GlyA), CD45, and CD133 and exposed to Aldefluor. UCB-derived total nucleated cells are visualized on a dot plot based on forward scatter (FS: x-axis) vs side scatter (SS; y-axis) signals. Panel 2: Cells from region R1 (panel 1) are further analyzed for CD45⁻/GlyA⁻ (region R2) and CD45⁺/Gly⁻ (region R7) populations. Panel 3: Cells from region R2 (panel 2) are sorted as CD45⁻/GlyA⁻/CD133⁺/ALDH^low (region R3) and CD45⁻/GlyA⁻/CD133⁺/ALDH^high (region R4) subfractions of VSELs. Panel 4: Cells from region R7 (panel 2) are sorted as CD45⁺/GlyA⁻/CD133⁺/ALDH^low (region R8) and CD45⁻/GlyA⁻/CD133⁺/ALDH^high (region R9) HSC subpopulations.

Fig. 9.3

Murine VSELs deposited in adult tissues are protected from excessive insulin/insulin-like growth factor signaling (IIS). (A) VSELs (green) are deposited in adult tissues as a backup population for tissue-committed stem cells, and express non-signaling insulin-like growth factor-2 receptor (IGF2R) and two signaling receptors, insulin like-growth factor-1 receptor (IGF1R) and insulin receptor (InsR). Thus, they can be stimulated by an autocrine IGF2 loop (left panel) and by paracrine/endocrine IGF-1 and insulin (right panel). (B) Due to changes in expression of paternally imprinted genes (e.g., Igf2R, Igf2, and RasGrf1), VSELs are protected from IIS, which would otherwise lead to their premature depletion from adult tissues as well as trigger uncontrolled proliferation and teratoma formation. Left panel: VSELs are protected from autocrine IIS by changes in expression of imprinted genes that are important in IIS. Down regulation of autocrine expression of IGF2, up regulation of IGF2R (a non-signaling receptor that binds IGF2 and prevents its binding to signaling IGF1R), and down regulation of RasGRF1 (which is involved in signal transduction from IGF2R), make VSELs less sensitive to autocrine/paracrine IGF2 signaling. Right panel: Down regulation of RasGRF1, which is involved in signaling from activated IGF1R and InsR in VSELs, also plays an important role in attenuation of IIS signaling by paracrine circulating levels of IGF1 and insulin. For the sake of simplicity, other imprinted genes (H19 and p57^Kip2) that are not involved in IIS and negatively affect VSEL proliferation are not depicted.