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. Author manuscript; available in PMC: 2010 Jun 5.
Published in final edited form as: Cell Stem Cell. 2009 May 7;4(6):513–524. doi: 10.1016/j.stem.2009.04.015

Figure 6. Resetting the identity of pluripotent states.

Figure 6

(A) 129 EpiSC cells derived from the epiblast of day E5.5 mouse embryos were reprogrammed into ICM-like cells after infection with TetO-Klf4 or c-Myc lentiviruses and culture in the mESM + Dox. Images on the bottom show representative colony formation observed 4–6 days after infection, which were passaged on MEF feeders. The cell lines were termed Epi-iPS. (B–C) Evaluation of Oct4 enhancer activity and sensitivity to JAKi and ALKi on the converted cell lines was performed as indicated in (Figure 4E–F). (D) Chimeric mice generated from the indicated Epi-iPS cells lines as evident by the agouti coat color. (E) Efficiencies of iPS derivation from EpiSC and somatic cells: 129 EpiSCs were infected with Teto-Klf4-2A-mOrange or Teto-cMyc-2A-mOrange lentiviruses and after 3 days of Dox inductions mOrange+ infected cells were single cell sorted in 96 well plates. Different hematopoietic cells from transgenic reprogrammable mice carrying the Dox inducible OSKM transgenes were single cell sorted in 96 well plates. HSC- hematopoietic stem cell enriched fraction, CMP- common myeloid progenitors. Efficiency for Nanog+ cells stable in mESM was determined at day 25 for all samples. Average results from 2 independent experiments are shown. *P value < 0.05 for the outlined efficiency comparisons. (F) Converting NOD EpiSC-like ES cells into transgene dependent ICM-like cells. EpiSC like cells were generated from NOD-ES line #43 after withdrawal of KP/CH and growing the cells in epiESM. As in (A) the line was infected with TetO-K and within 2 passages in mESM in the presence of Dox. Stable lines termed Epi-iPS were derived. The lower pictures indicate that the cell line required Dox or the presence of KP/CH to remain stable in culture. Factors and growth conditions utilized to facilitate the identity conversion at each step are indicated. The pluripotency state was evaluated and defined based on Oct4 enhancer activity and ALKi or JAKi sensitivity (indicated in blue or red).