Reprogramming primordial germ cells into pluripotent stem cells

Abstract

Background: Specification of primordial germ cells (PGCs) results in the conversion of pluripotent epiblast cells into monopotent germ cell lineage. Blimp1/Prmt5 complex plays a critical role in the specification and maintenance of the early germ cell lineage. However, PGCs can be induced to dedifferentiate back to a pluripotent state as embryonic germ (EG) cells when exposed to exogenous signaling molecules, FGF-2, LIF and SCF.

Methodology and principal findings: Here we show that Trichostatin A (TSA), an inhibitor of histone deacetylases, is a highly potent agent that can replace FGF-2 to induce dedifferentiation of PGCs into EG cells. A key early event during dedifferentiation of PGCs in response to FGF-2 or TSA is the down-regulation of Blimp1, which reverses and apparently relieves the cell fate restriction imposed by it. Notably, the targets of Blimp1, which include c-Myc and Klf-4, which represent two of the key factors known to promote reprogramming of somatic cells to pluripotent state, are up-regulated. We also found early activation of the LIF/Stat-3 signaling pathway with the translocation of Stat-3 into the nucleus. By contrast, while Prmt5 is retained in EG cells, it translocates from the nucleus to the cytoplasm where it probably has an independent role in regulating pluripotency.

Conclusions/significance: We propose that dedifferentiation of PGCs into EG cells may provide significant mechanistic insights on early events associated with reprogramming of committed cells to a pluripotent state.

Figure 1. Expression of Blimp1 and Prmt5 during PGCs reprogramming to EG cells.

(A) Schematic depicting dedifferentiation of PGCs to EG cells in the presence of LIF, FGF-2 and SCF. PGCs undergo reprogramming resulting in a large colony of pluripotent EG cells after 10 days of culture. Down-regulation of Blimp1 repressive complex may be a key early event leading to the generation of EG cells. (B) Expression of Blimp1 and Prmt5 in cultured PGCs. Oct4 is a marker of PGCs (red). Blimp1 (green) was detected on day 1 (labelled as 1d) of culture but not on 2d (dashed line). Prmt5 (green) was detected on day 1–4 in the nuclei of PGCs, but subsequently translocated to the cytoplasm on day 7 of culture. Merged images are shown with DNA stained with Toto-3 (blue). Scale bar, 30 µm. (C) The fate of Blimp1/Prmt5 complex during PGCs dedifferentiation to EG cells is depicted schematically.

Figure 2. c-Myc and Stat-3 expression is up-regulated in cultured PGCs.

PGCs were cultured in FGF-2, LIF and SCF. Oct-4 is a marker of PGCs (red). (A) Immunofluorescence staining of c-Myc (green) showed no expression of c-Myc during first 5-days of culture (dashed line), but the protein was detected on 7d in colonies consisting of 50–60 cells. (B) Immunofluorescence staining of Stat-3 (green). Stat-3 was not detected in 1d of PGCs culture (dashed line), but became detectable at 4d of culture mostly in the cytoplasm (dashed line), although we also observed small colonies of cells in which Stat-3 was nuclear (white line). Merged images are shown with DNA stained with Toto-3 (blue). Scale bar, 30 µm in B–E and G, scale bar 60 µm in F and H. (C) Diagram depicts LIF requirement in the reprogramming process. LIF was added to PGC after 1-, 2- or 4-days of culture. PGCs cultured in SCF, LIF and FGF-2 were used as control. After 10 days all cultures were stained for TNAP activity and the number of colonies was counted. We observed similar number of colonies in control and cultures in which LIF was added after 1-day (+++). Other cultures generated only one colony when LIF was added after 2-days, and no colonies formed when LIF added after 4-days.

Figure 3. TSA replaces FGF-2 in the reprogramming process.

(A) Approximately 80 PGCs from E 8.5 embryos were cultured in the presence of SCF, LIF and treated with TSA, at 5 or 15 ng/ml. In some cultures TSA (5 ng/ml) was added after 24-hours. PGCs cultured in SCF, LIF and FGF-2 were used as a control. After 10 days, cells were stained for TNAP activity and the number of colonies was counted. Graph shows combined results from three independent experiments for each TSA concentration. Error bars indicate standard deviation. Cultures treated with TSA (5 ng/ml) were reprogrammed more efficiently than PGCs in the control group, or cultures treated with TSA 24 hours later. TSA at 15 ng/ml was toxic to cells. (B) Immunofluorescence of TSA-treated cultures. Blimp1 (green) was down-regulated more rapidly compared to the control on 1d and was undetectable in 2d cultures. c-Myc (green) was detected in small colonies 5d cultures indicating an acceleration of the reprogramming process. Merged images are shown with DNA stained with Toto-3 (blue). Scale bar, 30 µm. (C) Model illustrating key events in the PGCs reprogramming to EG cells in the presence of FGF-2. Coloured boxes depict expression pattern of studied proteins. On day 1, cultured PGCs are in a repressive state due to expression of Blimp1/Prmt5 complex in nuclei of all PGCs. On day 2, PGC are released from lineage repression through the loss of Blimp1 (a brown box) and undergo further phenotypic changes leading to pluripotency when targets of Blimp1 such as Dhx38, c-Myc and Klf-4 are upregulated. On day 4 Stat-3 (a red box) translocates from cytoplasm-to-nuclei. Klf-4 is detected at low levels. On day 7 some cultured PGCs form large colonies. Cells of these colonies express c-Myc, while Prmt5 translocates from nuclei to the cytoplasm. Expression of Klf-4 and Dhx38 is at high levels in nascent EG cells. PGCs express Oct-4 and Sox2 throughout the culture period. The process is more efficient and is accelerated by 1–2 days when FGF-2 is replaced with TSA.