Abstract
Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.
Highlights
The ability to direct Embryonic stem (ES) and induced pluripotent stem cell differentiation toward specific tissue fates in vitro provides an excellent opportunity to investigate the gene regulatory networks (GRNs) that operate during organ development [1, 2]
Similar to other ES cell lines, cells in Pax6-GFP mES colonies displayed alkaline phosphatase activity, and immunostaining results were verified by RTPCR detection of Oct4 and Nanog expression in two independent Pax6-GFP mES cell line clones (Fig. 1H–I)
In this study we demonstrated that expression of either Pax6 or Six3 is sufficient to initiate lens marker expression and lentoid formation in differentiating mouse and human ES cells
Summary
The ability to direct ES and induced pluripotent stem (iPS) cell differentiation toward specific tissue fates in vitro provides an excellent opportunity to investigate the gene regulatory networks (GRNs) that operate during organ development [1, 2]. Markers, Prox, aB-crystallin, and Tdrd expression by immunostaining in these cultures at this stage whereas control vectors (LvHPV422 and LvHPV570) and vectors encoding either of two other genes, Eya, Ctnnb (encoding b-catenin), gave negligible staining (Fig. 3B–D,F, and data not shown). These data suggest that Pax and Six can induce markers of lens fiber cell fate in mES cells. Expression of either Pax or Six induces the expression of lens fiber cell markers
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