Manipulating cell fate in mouse embryos and embryonic stem cells

Complex signaling networks control all biological processes, including embryogenesis and maintenance of pluripotency in embryonic stem cells (ESCs). Feedbacks and cross talks between transcriptional factors in the network regulate the lineage segregation events during pre-implantation development as well as ESC pluripotency. Small molecules can activate or inhibit certain signaling pathway(s), thereby modulating the fate decisions of these cells. Therefore, we investigated the role of small molecules in lineage segregation in mouse embryos and subsequently in mouse ESCs (mESCs) pluripotency. We cultured mouse pre-implantation embryos in presence of small molecules and growth factors known to support the pluripotency in ESCs to evaluate their effect on lineage segregation. We found that inhibition of TGFβ signaling during mouse pre-implantation embryo development increased the number of NANOG positive epiblast cells, whereas its activation has the opposite effect. Next, we verified whether the increased Nanog positive cells in embryos help improve the mESC derivation from the embryos treated with an inhibitor of TGFβ signaling. We found that the ESC derivation efficiency from mouse embryos cultured in SB was significantly higher compared to control embryos. In addition, we also observed that inhibition of TGFβ signaling supports the ground state of pluripotency by significantly upregulating the expression of pluripotency makers in mESCs cultured in presence of its inhibitor. We also found an altered efficiency of cells to differentiate to endodermal and mesodermal lineage by culturing the mESCs in presence or absence of TGFβ pathway inhibitor before application of the differentiation protocol. We show that inhibition of TGFβ pathway improves the epiblast proliferation and subsequent ESC derivation efficiency in mouse models. Inhibiting the same pathway can alter the pluripotent state as well as differentiation of ground state mESCs. Inhibition of ROCK signaling during mouse embryo culture impairs blastocyst formation by suppressing trophectoderm (TE) cell proliferation and preventing the formation of blastocoel cavity. However, whether these embryos are capable of giving rise to mESCs or not, have not yet been reported. Therefore, we cultured mouse embryos in the presence of ROCK inhibitor (ROCKi, Y27632) to see how it affects the first lineage segregation events and eventual stem cell derivation. We found that with lower concentrations of ROCKi, the TE formation was significantly reduced without affecting the blastocysts formation rate and subsequent ESC derivation efficiency. However, when cells were cultured in higher concentrations of ROCKi, we found that embryonic developmental potential was severely compromised. Most of the embryos cultured in higher concentrations of ROCKi not only failed to form blastocysts on day 5 of in vitro culture. Despite these embryos exposed in higher concentrations of ROCKi were not able to yield blastocysts, they were still capable of giving rise to mESCs, however with low ESC derivation efficiency. During recent years, it became clear that different states of pluripotency exist in the mouse, namely naive, primed and ground state of pluripotency that are characterized by a unique transcriptional and epigenetic profiles. ESCs in naive state are in more pristine stage of pluripotency with lower expression of lineage specific markers whereas primed ESCs are already pre-disposed to spontaneous differentiation with higher expression of lineage specific markers. Ground state mESCs show improved naive state with closer resemblance to pre-implantation stage epiblast at (epi)genomic level. There is also a general consensus that the genetic background of mice affect stem cell derivation potential, as initial conditions of LIF/serum on mouse feeder layers only allowed successful mESC derivation in a limited number of mouse strains, particularly the 129 strain. Therefore, we compared transcriptional profiles of different states of pluripotent mouse stem cells (ground state, naive, primed) from the same mice strain to avoid genetic variation between the ESC lines, which has never been done thus far. For this, we performed genome-wide transcriptional analysis using microarrays on naive, primed and ground state mESCs. Our results showed that the primed state of pluripotency (shown by mouse epiblast stem cells, mEpiSCs) were transcriptionally very different from the naive (shown by mESCs in LIF+serum) and ground state ESCs (shown by mESCs in 2i). Naive and ground states of pluripotency in mESCs are closer but not similar to each other. The differentiation ability of these pluripotent stem cells towards different germ layers may thus vary depending on the used culture condition during derivation