CD133 (Prominin) Negative Human Neural Stem Cells Are Clonogenic and Tripotent

Yirui Sun,Steve Pollard,Liangfu Zhou,Jin Hu,Weiqing Kong,Austin Smith,Anna Falk,Alain Chédotal

doi:10.1371/journal.pone.0005498

Abstract

BackgroundCD133 (Prominin) is widely used as a marker for the identification and isolation of neural precursor cells from normal brain or tumor tissue. However, the assumption that CD133 is expressed constitutively in neural precursor cells has not been examined.Methodology/Principal FindingsIn this study, we demonstrate that CD133 and a second marker CD15 are expressed heterogeneously in uniformly undifferentiated human neural stem (NS) cell cultures. After fractionation by flow cytometry, clonogenic tripotent cells are found in populations negative or positive for either marker. We further show that CD133 is down-regulated at the mRNA level in cells lacking CD133 immunoreactivity. Cell cycle profiling reveals that CD133 negative cells largely reside in G1/G0, while CD133 positive cells are predominantly in S, G2, or M phase. A similar pattern is apparent in mouse NS cell lines. Compared to mouse NS cells, however, human NS cell cultures harbour an increased proportion of CD133 negative cells and display a longer doubling time. This may in part reflect a sub-population of slow- or non-cycling cells amongst human NS cells because we find that around 5% of cells do not take up BrdU over a 14-day labelling period. Non-proliferating NS cells remain undifferentiated and at least some of them are capable of re-entry into the cell cycle and subsequent continuous expansion.ConclusionsThe finding that a significant fraction of clonogenic neural stem cells lack the established markers CD133 and CD15, and that some of these cells may be dormant or slow-cycling, has implications for approaches to identify and isolate neural stem cells and brain cancer stem cells. Our data also suggest the possibility that CD133 may be specifically down-regulated during G0/G1, and this should be considered when this marker is used to identify and isolate other tissue and cancer stem cells.

Highlights

Findings of continuous neurogenesis in the mammal central nervous system (CNS) have raised great interest in neural stem and progenitor cells in both basic and applied neurobiology [1,2,3,4,5]
Our data suggest the possibility that CD133 may be downregulated during G0/G1, and this should be considered when this marker is used to identify and isolate other tissue and cancer stem cells
To determine whether CD133 and CD15 are expressed by human neural stem cells in culture, we performed immunostaining on three independent human NS cell lines, CB541, CB660, and CB660sp [26]

Summary

Introduction

Findings of continuous neurogenesis in the mammal central nervous system (CNS) have raised great interest in neural stem and progenitor cells in both basic and applied neurobiology [1,2,3,4,5]. CD133 immunopurification was later applied by Lee et al and Corti et al to isolate neurosphere forming precursor cells from mouse foetal cerebellum and forebrain [17,18]. In each of these cases, enriched cells expressed neural stem cell markers and were capable of multi-lineage differentiation both in vitro and in vivo [16,17,18]. CD133 expression could be detected in a relatively small subpopulation of cells in brain tumours [19] When these CD133+ cells were isolated, they were able to proliferate, form clonal neurospheres, and produce new tumors after serial transplantation [20,21,22]. The assumption that CD133 is expressed constitutively in neural precursor cells has not been examined

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Conclusion