Quiescent Status of Very Small Embryonic Like Stem Cells (VSELs) Points to Pivotal Role of Autocrine Role of Insulin-Like Growth Factor-2 (Igf2) – Ras-Activating Guanine Nucleotide Exchange Factor (Rasgrf1) Axis in Regulating Proliferation of Embryonic Stem Cells.

Abstract 1484

Poster Board I-507

The proper balance in expression of so-called imprinted genes is crucial for initiation of embryogenesis. Overall, there are ∼80 imprinted genes (expressed either from maternal or paternal chromosomes only), for which proper mono-allelic expression regulates totipotential status of the zygote and pluripotency of developmentally early stem cells. Of these 80 genes, only three (Igf2-H19, Rasgrf1, Meg3) are paternally methylated. We reported that the population of pluripotent VSELs deposited in adult organs (Leukemia 2006:20;857) is kept in quiescent status by erasure/demethylation of the paternal imprint on Igf2-H19 and Rasgrf1 loci and hypermethylation of the maternally imprinted Igf2R locus (Leukemia 2009, in press). Because of these epigenetic changes, VSELs do not express Igf2 or Rasgrf1, but overexpress non-signaling Igf2 receptor (Igf2R). It is known that while Igf2 is signaling through two tyrosine kinase receptors, i.e., insulin receptor (InsR) and insulin-like growth factor-1 receptor (Igf1R), Igf2R is a non-signaling surface glycoprotein that acts as a molecular sink to prevent binding of Igf2 to InsR or Igf1R. This suggests that Igf2 is an autocrine regulator of the early steps of embryogenesis. In addition, because VSELs do not express Igf2 but overexpress Igf2R, this causes a decrease in Igf2 signaling in these cells and explains their quiescence. Based on these observations, we hypothesized that the Igf2-Igf1R/InsR-Rasgrf1 axis is crucial for proliferation of the earliest embryonic stem cells. In fact, we found that Igf2, Insulin, Igf1R, InsR, and Igf2R are expressed in the established murine embryonic ES-D3 cell line. Moreover, ES-D3 cells express another Igf1R ligand, insulin-like growth factor-1 (Igf1). We also noticed that expression of Igf2 as well as Rasgrf1, but not Ins, Igf1, Igf1R, or InsR, is selectively and highly upregulated (∼100 times and ∼15 times, respectively) in cells isolated from rapidly expanding (for three days) ES-D3-derived embryonic bodies. More importantly, our Western blot data revealed that activated/phosphorylated Rasgrf1 is upregulated in ES-D3 cells after stimulation by Igf2, Igf1, and Insulin, suggesting this protein is involved in InsR and Igf1R signaling. To address this issue, we knocked down Rasgrf1 and Igf2 expression in ES-D3 cells employing an shRNA strategy. As such, downregulation of Rasgrf1 (by ∼80%) produced ES-D3 cells with significantly impaired proliferation that displayed profound defects in embryonic body growth in a hanging drop model as well as in leukemia inhibitory factor-depleted cultures. Furthermore, we found that Rasgrf1^-/− cells lost chemotactic responsiveness to all insulin factors. In contrast, knock down of Igf2 did not produce a significant phenotype, most likely because of redundancy or compensation by Igf1 and Ins. Conclusions. For the first time, our data provide novel evidence that insulin factors secreted by early embryonic cells are crucial for their proliferation/expansion and that Rasgrf1 is an important protein involved in signaling from activated Igf1R and InsR. The Igf2 - Igf1R/InsR - Rasgrf1 axis plays a crucial role in the proliferation of embryonic stem cells and is required for embryoid body formation. However, autocrine-secreted Igf1 and Ins could replace Igf2. Rasgrf1 seems to be an obligatory molecule to maintain proper signaling from InsR and Igf1R. Perturbation of these pathways by epigenetic changes in somatic-imprinted genes explains the quiescence of VSELs deposited in adult tissues. On the other hand, positive modulation of this axis is crucial for developing more powerful strategies to “unleash the power” of these cells and employ them in regenerative medicine.