FCMAB117P Sigma-AldrichAnti-SSEA-1 Antibody, clone MC-480, PE conjugate

Different interventions are being tested for restoration of the youthfulness of adult mouse-derived fibroblasts. However, fundamental issues, such as the decline of adult mouse-derived fibroblast activity with age, remain unresolved. Therefore, in this study, we examined whether treatment with collagen complexes has beneficial effects on the rejuvenation or reprogramming of adult mouse-derived fibroblasts. Further, we investigated the mechanisms of rejuvenation of adult mouse-derived fibroblasts during treatment with total collagen complexes. We isolated total collagen complexes from the tails of young mice and cultured adult mouse-derived fibroblasts with or without the collagen complexes. When compared with fibroblasts cultured without collagen complexes, adult-derived fibroblasts cultured with collagen complexes over five consecutive passages showed a more youthful state, expanded at a higher rate, and exhibited reduced spontaneous cell death. The fibroblasts cultured in the presence of collagen complexes also showed extensive demethylation in the promoter regions of cell cycle-related genes such as PCNA, increased proliferation, and decreased senescence. In addition, the efficiency of reprogramming of fibroblasts to become induced pluripotent stem (iPS) cells was significantly higher in young- and adult-derived fibroblasts cultured with collagen complexes than in adult-derived fibroblasts cultured alone. Furthermore, mechanistic evidence shows that genes involved in anti-proliferative pathways, including Ink4a/Arf locus genes and p53, were downregulated in fibroblasts exposed to collagen complexes. Interestingly, our results suggest that the rejuvenation process was mediated via the α2β1 integrin-dependent Bmi-1 pathway. Thus, collagen complexes both stimulate proliferation and inhibit cell death and growth arrest in fibroblasts, which appears to be a promising approach for improving the efficiency of reprogramming.

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 agreater than 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.