Our goal was to analyze the functional significance of hESC subpopulations, as defined by the expression of specific surface antigens. A fluorescence-tagged co-culture system allowed us to both determine the lineage fate of differentiating hESC subpopulations and characterize the cell surface profile of proliferating hESC subpopulations in vitro.
CD135 (Flt3) is a tyrosine kinase receptor that has been associated with tissues derived from mesoderm, including hematopoietic stem cells, myelomonocytic progenitors, primitive B-cell progenitors, and thymocytes [16
]. CD133 (prominin-1) is a member of a class of novel pentaspan membrane proteins identified in both humans and mice. Although classified as a marker of primitive hematopoietic and neural stem cells [17
], tissue expression arrays have shown that human CD133 mRNA is strongly expressed in differentiated tissues derived from all three embryonic germ layers, including kidney, mammary gland, trachea, pancreas, digestive tract, and testis [18
]. A population of CD133+
cells isolated from the adult human kidney was capable of both self-renewal and multilineage differentiation in vitro and in vivo [19
]. CD133 is also expressed on endothelial progenitor cells, which play a role in angiogenesis and neovasculogenesis during both tumor growth and wound healing [20
subpopulations, isolated from developing mouse brain, were capable of clonal expansion to form neurospheres in vitro and differentiated into multiple lineages in vitro and in vivo following transplantation into neonatal mice [21
]. Although CD133 is expressed on a broad range of adult tissues, the significance of its expression on undifferentiated hESCs has previously not been understood.
Both CD133+GFP+ and CD133−GFP+ selected cells displayed the ability to efficiently self-renew in a co-culture with H9 GFP−hESCs. Differences between these two subpopulations became evident upon differentiation in vitro. The vast majority (~90%) of the CD133+GFP+ subpopulation followed an ectodermal lineage fate. This contrasted sharply with differentiated CD133−GFP+ hESCs, which predominantly stained for endodermal and mesodermal markers. The ability of CD133+GFP+ hESCs to follow an ectodermal lineage fate appears to be CD133-specific, since CD135+GFP+ hESCs differentiate into all three somatic lineages similar to wild-type H9 hESCs. These results suggest that a CD133+ subpopulation of proliferating hESCs are predisposed to follow an ectodermal lineage fate. CD133+ hESCs may indeed represent one of many subpopulations of progenitor cells within an undifferentiated hESC colony, which can self-renew and follow a preprogrammed, preferential lineage fate.
Previous studies have demonstrated the association of the CD133 surface antigen with embryonic neural stem cells in developing mouse embryos [22
]. After culture in vitro, CD133−
cells isolated from embryonic mouse brains failed to form neurospheres, while CD133+
cells formed neurospheres, gave rise to astrocytes and neuronal cells, and expressed nestin [24
]. Another study used an in vitro system to follow the expression CD133 on differentiating mouse embryonic stem cells (mESCs) [25
]. CD133 expression was associated with proliferating mESCs, and its expression was maintained during the early stages of differentiation into ectodermal, endodermal, and mesodermal precursor cells. At the terminal stages of differentiation, however, expression of CD133 was observed only on cells co-expressing the neuroectodermal marker, nestin [25
]. In human studies, CD133+
cells derived from human brain tumors have been shown to display the properties of self-renewal and recapitulation of the original tumor, suggesting that CD133 may mark cancer stem cells specific to neural tumors [26
]. Together, these studies provide further evidence to suggest a role for CD133 as a lineage-specific antigen for neural precursor cells in mice and humans.
We also wanted to determine whether the expression of a lineage-confirmed surface marker was maintained over time within proliferating cultures. To address this issue, we monitored the expression of a set of surface antigens during the expansion of selected hESCs in the fluorescence-tagged co-culture system. We discovered that both the CD133+GFP+ and CD135+GFP+ subpopulations continued to express surface antigens CD133, CD135, FGFR-1, SSEA-4, and Tra-1-60, following multiple passages. The amounts of these surface antigens remained constant as compared to the levels of the same antigens detected on zero-passage, unsorted GFP+ hESCs. These results support the concept that stable subpopulations of hESCs exist within proliferating cultures. More importantly, these subpopulations can be expanded without the loss of marker expression or lineage specificity.
We observed that the percentage of CD133+
hESCs co-expressing the pluripotency antigen, Tra-1-60, as well as CD135 and FGFR-1, was similar to that of the unsorted cells. In contrast, over 90% of the CD133+
hESCs were found to co-express SSEA-4, whereas SSEA-4 was only detected on 70% of CD133−
cells. This suggested that SSEA-4 preferentially co-localizes with CD133 in proliferating hESC cultures. A previous study has also demonstrated that greater than 90% of CD133+
cells co-express SSEA-4 in a proliferating H9 hESC culture [2
]. A recent report suggests a link between CD133 co-expression with SSEA-4 and the positive selection of human neural progenitor cells. SSEA-4 was found to be expressed during human central nervous system development in 6–9-week-old human embryos, and the selection of cells from embryonic forebrains, expressing both SSEA-4 and CD133, resulted in the enrichment for neural progenitor cells [29
]. The SSEA4+
subpopulation was expanded in a neurosphere culture, and the SSEA4+
phenotype was retained after several passages [29
Our data demonstrate that subpopulations characterized by the expression of distinct surface markers exist within proliferating hESC cultures in vitro. Importantly, this suggests that the concept of “pluripotency” within proliferating hESC cultures may not apply to all cells within the culture. We were able to efficiently expand these selected subpopulations using a fluorescence-tagged co-culture system, and demonstrate that marker expression of the selected hESCs was retained during expansion. The majority of CD133-selected hESCs, differentiated by embryoid body formation and by teratoma formation, followed a neuroectodermal lineage fate. This suggests that CD133 will be useful as a marker for the in vitro expansion of hESCs that will give rise specifically to neural progenitor cells for the potential treatment of neurodegenerative and neurotraumatic disorders.