Embryonic germ cell extracts erase imprinted genes and improve the efficiency of induced pluripotent stem cells

Abstract

Patient-specific induced pluripotent stem cells (iPSCs) have the potential to be useful in the treatment of human diseases. While prior studies have reported multiple methods to generate iPSCs, DNA methylation continues to limit the totipotency and reprogramming efficiency of iPSCs. Here, we first show the competency of embryonic germ cells (EGCs) as a reprogramming catalyst capable of effectively promoting reprogramming induced by four defined factors, including Oct4, Sox2, Klf4 and c-Myc. Combining EGC extracts with these four factors resulted in formation of more embryonic stem cell-like colonies than did factors alone. Notably, expression of imprinted genes was higher with combined induction than with factors alone. Moreover, iPSCs derived from the combined inductors tended to have more global hypomethylation. Our research not only provides evidence that EGC extracts could activate DNA demethylation and reprogram imprinted genes, but also establishes a new way to enhance reprogramming of iPSCs, which remains a critical safety concern for potential use of iPSCs in regenerative medicine.

Figure 1

Treatment with embryonic germ cell (EGC) extracts enhances the generation of induced pluripotent stem cells (iPSCs). (A) Schematic representation of iPSC generation from mouse embryonic fibroblasts (MEFs) by ectopic expression of Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by exposure to EGC extracts. (B) Sequential morphology of iPSC generation with EGC extract treatments (a) of MEFs (b–d) Morphology of epithelium-like cells after infection at day 3 (e) embryonic stem cell-like colonies formed at day 5 (f) and as colonies become bigger at day 7. (C) Left: Cell cycle by FACS at day 3 and 6 from four factor-infected MEFs treated with EGC extracts and four factor-infected MEFs. Right: Percentage of cells in each cell cycle phase and PI (cell cycle proliferation index) in OSKM transfected MEFs with or without EGC extracts treatment. Mean values ± standard error of the mean (SEM) of three independent experiments are shown. (D) Left: Representative images of alkaline phosphatase-positive (AKP + ) colonies. Cells were fixed at day 8. Right: Number of AKP + colonies. Mean values ± SEM of a representative experiment are shown, n = 3. Scale bar = 100 µm.

Figure 2

Expression of pluripotent genes, In vitro and in vivo differentiation and the of iPSCs generated by EGC extract and OSKM combination. (A-a) Morphology of EG-4F-iPSCs P15. (A-b) Alkaline phosphatase-positive colonies and (A-c) normal karyotype (20pairs). (B) Quantitative PCR analysis of pluripotent markers Oct4, Nanog, Rex1 and Sox2 in EG-4F-iPSCs. Error bars indicate standard error of the mean (n = 3). Results were normalized to GAPDH expression. *P < 0.01, versus other cells. (C-a) Embryoid body formation by suspension culture. (C-b) Teratomas. (C-c) RT-PCR analysis of germ layer markers. The grouping blots of germ layer markers were cropped from different parts of the same gel. The grouping blots of gapdh were cropped from different parts of the same gel. (D) Hematoxylin and eosin staining of teratomas. Scale bar = 50 µm. (E) Immunofluorescence staining of pluripotent genes.

Figure 3

Genomic methylation patterns. (A) Hypo- and hypermethylation peak counts obtained from three cell lines in each group. EG-4F-iPSC versus 4F-iPSC peaks were considered as hypermethylation peaks; 4F-iPSC versus EG-4F-iPSC peaks were considered as hypomethylation peaks. (B) Average proportions of peaks within each region as defined by genomic structure. *P < 0.05 (C) Average proportions of peaks within each region as defined by distance from the CpG island. (D) Enrichment analysis of hypomethylated genes covered with peaks. (E) Comparison of imprinted gene expression in EG-4F-iPSCs and 4F-iPSCs as quantified by mRNA expression. Error bars indicate standard error of the mean (n = 3). Results were normalized to human histone H2A.Z (H2AFZ) expression. (F) Bisulfite sequencing of Nanog and OCT4 promoter region. Black circles represent methylated sites, white circles represent unmethylated sites. Global methylated cytosines are shown as %M.

Figure 4

Reprogramming of permeabilized MEFs induced by EGC extracts. (A) Proliferation rate of EGC extract-treated MEFs compared with MEF extract-treated MEFs at different days. Error bars represent standard error of the mean (SEM; n = 3). (B a–d) Morphology of colonies formed following treatment of MEFs with EGC extracts. (C) Induction of alkaline phosphatase activity in MEFs after exposure to EGC extracts. (D a-b) Immunofluorescence staining of Oct4 positive colonies following exposure of MEFs to EGC extracts. Scale bars = 25 µm. (E) Quantitative PCR analysis of pluripotency marker and laminA expression in MEFs after incubation with EGC extracts. Error bars indicate SEM (n = 3). Results were normalized to GAPDH expression. *P < 0.05, versus control. (F) Q-PCR analysis of imprinted gene expression between EGC extract-treated MEFs and MEF extract-treated MEFs at 4 days after treatment. Results were normalized to human histone H2A.Z (H2AFZ) expression. *P < 0.05, **P < 0.01, versus control.