FGF4 And its Novel Splice Isoform Have Opposing Effects on the Maintenance of Human Embryonic Stem Cell Self Renewal

¹ Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University, Jerusalem, Israel
² ProChon Biotech Ltd; Golda Meir St., Weizmann Science Park, Nes Ziona 70400, Israel

^* To whom correspondence should be addressed. E-mail: nissimb{at}mail.ls.huji.ac.il.

	Abstract

Human embryonic stem cells (HESCs) are unique in their capacity to self renew while remaining pluripotent. This undifferentiated state must be actively maintained by secreted factors. In order to identify autocrine factors that may support HESC growth we have taken a global genetic approach. Microarray analysis identified FGF4 as a prime candidate for autocrine signaling. Furthermore, the addition of recombinant FGF4 to HESCs supports their proliferation. We show that FGF4 is produced by multiple undifferentiated HESC lines along with a novel FGF4 splice isoform (FGF4si) that codes for the amino-terminal half of FGF4. Strikingly, while FGF4 supports the undifferentiated growth of HESCs, FGF4si effectively counters its effect. We further show that FGF4si is an antagonist of FGF4, shutting down FGF4 induced Erk1/2 phosphorylation. Expression analysis shows that both isoforms are expressed in HESCs and early differentiated cells. However, while FGF4 ceases to be expressed in mature differentiated cells, FGF4si continues to be expressed after cell differentiation. Targeted knock down of FGF4 using siRNA increased differentiation of HESCs, demonstrating the importance of endogenous FGF4 signaling in maintaining their pluripotency. Taken together these results suggest a growth-promoting role for FGF4 in HESCs and a putative feedback inhibition mechanism by a novel FGF4 splice isoform that may serve to promote differentiation at later stages of development.