Cbx7 promotes the generation of induced pluripotent stem cells

Abstract

The iPS cells were discovered in 2006. With their ability to differentiate into cells of all three germ layers, iPS cells have great potential for clinical applications. Oct4, Sox2, c-Myc, and Klf4 were identified as the most effective factors for generating iPS cells. Despite this, iPS cells manufactured with these factors would still be inefficient. As a member of the chromobox family, chromobox protein homolog 7 (Cbx7) binds to PRC1 and PRC2 to inhibit genes involved in differentiation. A decrease in the expression of Cbx7 is observed during embryonic stem cell differentiation. Currently, no report discusses the role of Cbx7 in the production of iPS cells. In this study, we hypothesized that Cbx7 could increase iPS cell generation. We confirmed that Cbx7 is highly expressed in pluripotent stem cells (including ES and iPS cells). In addition, transfecting Cbx7 into fibroblasts increased Oct4, Sox2, c-Myc, and Klf4 expression. Moreover, we describe a novel approach to producing iPS cells using Cbx7 in combination with Oct4, Sox2, c-Myc, and Klf4. In summary, we have demonstrated that Cbx7 enhances the reprogramming of iPS cells and characterized the stemness and pluripotency of iPS cells.

Keywords: Cbx7; Induced pluripotent stem cells; Yamanaka factors.

Fig. 1

The expression levels of Cbx7 and SSEA1 in MEF, iPSCs, and ESCs. (A) Immunofluorescent staining showed that Cbx7 and SSEA1 were highly expressed in iPSCs and ESCs, but not in MEFs. Digital images were taken at a magnification of 40×. (B) The quantitative results showed Cbx7 and SSEA1 levels significantly higher in iPSCs and ESCs than in MEFs. Bars represented mean and SD. Differences between MEF and ESC or iPSC groups evaluated by two-tailed Student's t-test. ∗P < 0.05 indicates statistical significance (∗∗∗P < 0.001).

Fig. 2

Schematic illustration of the reprogramming protocol. (A) The reprogramming strategy of iPS-OKMS and iPS-OKMSC cells. (B) Comparison of the reprogramming efficiency of iPS-OKMS and iPS-OKMSC cells. The ES-like colonies were counted on day 30. Bars represented mean and SD. Differences between OKMS and OKMSC groups were evaluated by a two-tailed Student's t-test. ∗P < 0.05 indicates statistical significance.

Fig. 3

Real-time PCR analysis of the expression levels of Yamanaka factors in Cbx7-overexpressing MEFs. The expression of Gapdh was detected as the reference gene. Bars represent mean and SD. The difference between groups was evaluated by a two-tailed Student's t-test. ∗P < 0.05 indicates statistical significance. (∗P = 0.01–0.05; ∗∗P = 0.001–0.01).

Fig. 4

RT-PCR analysis of stem cell-related genes in MEFs, iPS-OKMS cells, iPS OKMSC cells, and mouse ESCs. The expression levels were normalized to the mES group. RT(-): real-time PCR negative control. The signal was quantified with ImageJ software.

Fig. 5

The alkaline phosphatase staining and immunofluorescent staining of iPS-OKMS and iPS-OKMSC cells. (A) The activities of alkaline phosphatase were detected by immunocytochemistry staining in iPS-OKMS and iPS-OKMSC cells. Digital images were taken at a magnification of 100×. (B) The stem cell markers (Oct4, Sox2, and SSEA1) were explored with immunofluorescent staining. Digital images were taken at a magnification of 200×.

Fig. 6

Pluripotency assessment of iPS-OKMS and iPS-OKMSC cells. (A) iPS-OKMS and iPS-OKMSC cells were grown into embryoid bodies and seeded onto 0.1% gelatin-coated cell culture plates containing a differentiation medium. The expression of markers specific for the three germ-layer in EB-outgrowth cells was visualized by immunofluorescent staining: Gata4 (endoderm marker), alpha-smooth muscle actin (alpha-SMA) (mesoderm marker), and Tuj-1 (ectoderm marker). Nuclei were stained with DAPI (blue). Digital images were taken by a fluorescent microscope at a magnification of 400×. (B) The stem cell pluripotency was evaluated by in vivo teratoma formation assay. HE staining results indicated that iPS-OKMS and iPS-OKMSC cells possessed the ability to differentiate into all the 3-germ layer cells. Digital images were taken at a magnification of 200×.