Investigation into germ cell fate determination of rat embryonic stem cells
Taylor, Ryan Mathew
The laboratory rat is an important experimental model in biomedical research. Rat embryonic stem cells (ESCs) are valuable for studying mammalian development and for facilitating germ line-modification of rat strains. However, the germline contribution of rat ESCs when injected into developing rat embryos can be variable, restricting their utility. This investigation addressed two key questions; could rat ESC differentiation in vitro be directed towards the germline, and could the proportion of cells entering the germ cell lineage be increased by manipulating the gene network of epiblast cells? The formation of the unipotent germline precursors known as primordial germ cells (PGCs) is induced by BMP4/WNT3-mediated induction of essential PGC transcription factors (TFs) BLIMP1, PRDM14 and AP2γ within the epiblast. These factors cooperate to direct a small subset of cells at the proximal posterior region of the epiblast towards a germ cell fate by re-activating expression of naïve pluripotency markers whilst upregulating PGC-specific transcription factors. To promote differentiation of rat ESCs into the PGC fate, cells were subjected to a differentiation protocol previously developed for mouse ES-to-PGC-like cell (PGCLC) differentiation, which was modified for rat ESCs. Using this protocol, a small proportion of rat cells expressing the PGC-associated surface marker CD61 exhibited enhanced expression of PGCLC gene markers, indicating that the differentiation protocol had generated a population of rat PGCLCs. To increase the efficiency of PGCLC differentiation, three strategies were implemented to manipulate the epiblast gene network to improve the proportion of rat ESCs entering the germline lineage. The first strategy involved the use of geneediting technology (CRISPR/Cas9), to incorporate a PGC gene cassette expressing a transgene copy of Blimp1 immediately downstream of the epiblast gene promotor of Brachyury. It was hypothesised that insertion of this gene cassette would drive expression of the Blimp1 transgene during the epiblast phase of rat ESC fate determination and improve the number of rat PGCLCs generated during PGCLC differentiation. Indeed, the proportion of cells expressing the PGC-associated surface marker CD61 was elevated compared to the parental unmodified cell line. Additionally, CD61+ve cells generated from cells containing the Blimp1 transgene displayed increased expression of early PGC markers. The second strategy involved the conditional expression of the three key PGC transcription factors, Blimp1, Prdm14 and Ap2γ from stably integrated doxycyclinedependant Tet-On transposon vectors. Stably transduced cells cultured in doxycycline during embryoid body and PGCLC differentiation protocols had a greater proportion of CD61+ve cells and increased PGC marker gene expression compared to the parental cell line. This was most noticeable in cells expressing Blimp1 and either of the other two PGC transcription factors (Prdm14 or Ap2γ). Finally, CRISPR/Cas9 gene editing was also used to inactivate the rat Otx2 transcription factor gene. Deletion of Otx2 in mouse ESCs has been shown to increase the proportion of PGCLCs generated during germline differentiation in vitro. Similar to exogenous PGC transcription factor expression, rat Otx2-/- knock-out cells generated a greater proportion of CD61+ve cells compared to the parental cell line when put through a PGCLC differentiation protocol. CD61+ve Otx2-/- cells also had increased PGC transcription factor expression compared to the parental cell line, suggesting the loss of Otx2 can help drive the expression Blimp1, Prdm14 and Ap2γ. In conclusion, rat ESCs subjected to a PGCLC differentiation protocol and manipulation of PGC transcription factor expression exhibited increased expression of PGC markers. However, this response was more limited than that obtained with mouse ESCs, a result which is consistent with reduced germ line transmission of rat ESCs in chimaeric rats. This difference could be due to the initial pluripotency state of rat ESCs; rat ESCs might be in a more committed primed state compared to naïve mouse ESCs, reducing their competence for germ cell lineage differentiation. Additionally, rat ESCs may require different combinations of signals or regulatory factors to ensure efficient germ cell differentiation in vitro. Further development of the cell lines and reagents established during this investigation should provide a better understanding of germ line differentiation in the rat.