Instructing naïve pluripotency : JAK/STAT3 and Nanog in reprogramming

• Main Author: Van Oosten, Anouk Lisanne
• Format: Dissertation
• Language: English
• Published: University of Cambridge 2013
• DOI: 10.17863/CAM.11778

Reprogramming of somatic cells to pluripotency is a topic of extensive research driven by the potential of induced pluripotent cells in in vitro disease modelling, drug screening and regenerative medicine. The pluripotency associated transcription factor Nanog has been subject to a lot of controversy. Initially thought to form an essential trinity with Oct4 and Sox2 to maintain embryonic stem cell (ES) self-renewal, it was shown that actually ES cells can maintain their pluripotency in the absence of Nanog. In vivo however, Nanog is essential to establish the pluripotent epiblast. For Nanog -/somatic cells reprogramming by defined factors, in a culture condition using inhibitors for MEK/ERK and GSK3 (2i) fails to occur. This led to the assumption that acquisition of pluripotency in reprogramming is dependent on Nanog. This observation however, had not been verified extensively in alternative culture conditions. Using neural stem cells as a system to study reprogramming, I investigated the use of Knockout Serum Replacement (KSR) + LIF medium instead and found that in this condition reprogramming of Nanog -/- cells in fact does occur, Nanog is thus not absolutely required for the induction of pluripotency. The addition of 2i to KSR +LIF medium abolished reprogramming of Nanog -/- cells again. I did observe that the efficiency of reprogramming by defined factors is enhanced by constitutive overexpression of Nanog. I also found that addition of constitutively overexpressed Nanog to the generally used reprogramming factors enables reprogramming of somatic cells in a culture condition that does not support pluripotency, however presence of LIF was required. LIF was dispensable for maintenance of the obtained Nanog overexpressing iPS cells, suggesting that it could play a role in the acquisition of pluripotency. I also observed that if LIF is added to 2i medium during reprogramming the number of iPS cell colonies that emerges is enhanced. LIF contributes to maintenance of pluripotency via activation of the JAK/ST AT3 signalling pathway. ST AT3 was found to reprogram post-implantation epiblast derived stem cells (EpiSCs) to nai:ve pluripotency. I showed that for reprogramming of adult neural stem cells, increased activation of JAK/ST AT3 enhances efficiency and acts as a potent reprogramming factor. In addition, I demonstrated that sufficient activation of JAK/ST AT3 can overcome the reprogramming block of pre-iPS cells and obviates a need for further pluripotency culture environment requisites for reprogramming and maintenance of nai:ve pluripotency. I showed that FGF signalling, which promotes the exit of nai:ve pluripotent cells from self-renewal, does not prevent JAK/ST A T3 driven EpiSC conversion to nai:ve pluripotency. Moreover, even in the presence of FGF plus Activin, which instructs and maintains the primed state, I found that JAK/ST AT3 can dominantly enforce a nai:ve pluripotent state to EpiSCs.