Reprogramming pig fetal fibroblasts reveals a functional LIF signaling pathway.

ABSTRACT

Distinct signaling pathways are reported to maintain pluripotency in embryo-derived stem cells. Mouse embryonic stem cells (ESCs) respond to leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-mediated activity, whereas human ESCs depend upon Fibroblast growth factor (FGF) and activin signaling. In the majority of mammals investigated, however, the signals that support stem cell pluripotency are not well defined, as is evident by the persistent difficulties in maintaining authentic stable ESC lines. Induction of pluripotency by transcription factor-mediated reprogramming could provide an alternative way to produce ESC-like cells from nonpermissive species, and facilitate identification of core ESC signaling requirements. To evaluate the effectiveness of this approach in pigs, we transduced porcine foetal fibroblasts with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc, and maintained the resulting cultures in medium containing either LIF or FGF2. Alkaline phosphatase positive colonies with compact, mouse ESC-like morphology were preferentially recovered using serum-free medium supplemented with LIF. These cell lines expressed the endogenous stem cell transcription factors, OCT4, NANOG, and SOX2, and the cell surface marker SSEA-4, consistent with acquisition of an undifferentiated state. However, restricted differentiation potential, and persistent expression of retroviral transgenes indicated that reprogramming was incomplete. Interestingly, LIF activated both the transcription factor STAT3 and its target gene SOCS3, and stimulated cell growth, indicating functional coupling of the signaling pathway in these cells. This demonstration of LIF-dependence in reprogrammed pig cells supports the notion that the connection between LIF/STAT3 signaling and the core regulatory network of pluripotent stem cells is a conserved pathway in mammals.