(453f) Stem Cell Pluripotent State Transition: Going From Embryonic Stem Cells to An Extraembryonic Precursor State and Back

Stem cells, in particular pluripotent stem cells, hold great potential for regenerative medicines. Their practical use in clinical applications will require a large and steady supply of cells. These pluripotent stem cells in culture are prone to spontaneous deviation from a high degree of homogeneity. Furthermore, direct transplant of pluripotent stem cells bears the risk of tumorigenicity. Because of these shortcomings, highly multipotent stem cells derived from pluripotent stem cells that still possess the differentiation capacity to the desired lineage may serve as a means of expanding cell mass for clinical applications. In many cases, the maintenance and expansion of those multipotent stem cells is more robust and amenable to scalable processes.

Multipotent adult progenitor cells (MAPCs) derived from rat bone marrow are one of the multipotent stem cells.  Their differentiation state appears to be equivalent to multipotent extraembryonic endoderm (ExEn) precursor (XEN-P) cells which can be derived from rodent blastocysts. Both cell types express pluripotency-associated transcription factor, Oct4, at high levels very close to that of rat embryonic stem cells (rESCs); however, unlike rESCs, they also express extraembryonic endoderm-specific genes including Gata6, Gata4, Pdgfra, and Sox7 at high levels, but lack Nanog and Sox2.

We uncovered trajectories that allow rESCs to transit to XEN-P state, and, with a restricted window of time, to revert back to ES state. When grown in the presence of PDGF, EGF, and LIF, rESCs undergo morphological and gene expression changes to become like MAPCs and XEN-P cells within 10 days. Interestingly, the ExEn precursor-like cells revert to ESCs when transferred to mouse embryonic fibroblast (MEF) feeder cells with supplements of two inhibitors (2i), MEK and GSK inhibitors, although only with a low frequency. The differentiated cells from rESCs express extraembyonic endoderm-specific markers, Gata6, Gata4, Pdgfra, Sox7, and Sox17 as highly as XEN-P cells and MAPCs without loss of Oct4 expression. Immunostaining results suggest that the expression of Nanog and Gata6 in the ExEn precursor-like cells is not mutually exclusive, even though their simultaneous expression in a cell is rarely reported and is generally thought to be mutually repressive of each other’s expression. We have constructed a mathematical model describing the network consisted of Oct4, Sox2, Nanog, and Gata6 that contribute to the regulation of pluripotency. The experimental ESC-XEN-P transition system allows the dynamics of this gene network to be studied through treatments with signaling molecules and genetic manipulations. This model system of pluripotent state transition is a valuable tool for better understanding of the underlying regulation of pluripotency and differentiation.