CD9 Is Associated with Leukemia Inhibitory Factor-mediated Maintenance of Embryonic Stem Cells

Abstract

Mouse embryonic stem (ES) cells can proliferate indefinitely in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), or differentiate into all three germ layers upon removal of this factor. To determine cellular factors associated with self-renewal of undifferentiated ES cells, we used polymerase chain reaction-assisted cDNA subtraction to screen genes that are expressed in undifferentiated ES cells and down-regulated after incubating these cells in a differentiation medium without LIF for 48 h. The mRNA expression of a tetraspanin transmembrane protein, CD9, was high in undifferentiated ES cells and decreased shortly after cell differentiation. An immunohistochemical analysis confirmed that plasma membrane-associated CD9 was expressed in undifferentiated ES cells but low in the differentiated cells. Addition of LIF to differentiating ES cells reinduced mRNA expression of CD9, and CD9 expression was accompanied with a reappearance of undifferentiated ES cells. Furthermore, activation of STAT3 induced the expression of CD9, indicating the LIF/STAT3 pathway is critical for maintaining CD9 expression. Finally, addition of anti-CD9 antibody blocked ES cell colony formation and reduced cell viability. These results indicate that CD9 may play a role in LIF-mediated maintenance of undifferentiated ES cells. INTRODUCTIONMouse embryonic stem (ES) cells, which originally derived from inner cell mass of an early embryo named blastocyst, are able to sustain their pluripotency in in vitro cell culture (Evans and Kaufman, 1981;Martin, 1981). Undifferentiated mouse ES cells can be maintained for a long time in media containing the cytokine leukemia inhibitory factor (LIF) (Smith et al., 1988;Williams et al., 1988). Pluripotency of such cultured ES cells has been demonstrated in both in vivo and in vitro experiments. When injected into blastocysts, ES cells participate in embryonic development involving all three germ layers producing chimeric mice (Bradley et al., 1984). ES cells also form teratomas containing various mature tissues when injected into immunocompromised mice (Evans and Kaufman, 1981;Martin, 1981). In vitro, mouse ES cells start to differentiate to every possible lineage upon removal of LIF from the culture medium. The mechanism by which LIF maintains ES cells in undifferentiated state is not completely understood. The transcription factor STAT3 is a downstream target of LIF and its receptor interaction Matsuda et al., 1999). It has been shown that the activity of STAT3 is necessary and sufficient for LIF-induced self-renewal of mouse ES cells (Matsuda et al., 1999). It remains unclear, however, which genes are regulated by the STAT3 transcription factor in mouse ES cells and play actual roles in the maintenance of stem cells. Indeed, there is no generally accepted mechanism by which stem cells are maintained as undifferentiated cells. Human ES cell cultures have been recently established using mouse fibroblasts as feeder cells (Shamblott et al., 1998;Thomson et al., 1998). In contrast ...

Basic Fibroblast Growth Factor Supports Undifferentiated Human Embryonic Stem Cell Growth Without Conditioned Medium

“…3). In addition, in all the cultures maintained in bFGF and growth factors, most of the SSEA4 hi cells expressed TRA-1-60 [1,2,5,48] and CD9, a tetraspan transmembrane protein [49], another surface marker that is highly expressed in undifferentiated hESCs [4] (Fig. 3).…”

Establishment of a new embryonic stem cell line derived from C57BL/6 mouse expressing EGFP ubiquitously

“…We also performed RT-PCR assays to analyze expression of Pou5f1, the activity of which is essential for the identity of the pluripotent cell population in mammalian embryos (Nicols et al, 1998), and Pecam1, Utf1, Cd9, Zfp42, and Spp1, which are expressed in undifferentiated ES cells (Botquin et al, 1998;Hosler et al, 1989;Oka et al, 2002;Okuda et al, 1998;Robson et al, 2001). As shown in Figure 2a, alkaline phosphatase activity was present in colonies composed of ES-like cells on a feeder layer with no activity (Fig.…”

Claudin 6: a novel surface marker for characterizing mouse pluripotent stem cells

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