Figure 1.
Generation of FGF-iPS from mouse fibroblasts by retroviral transduction.
Schematic diagram illustrating a timetable of the critical steps of the reprogramming protocol utilized. Embryonic mouse fibroblasts were transduced with retroviruses carrying Oct4, Sox2 Klf4 and c-Myc and then transferred onto MEF feeder layers in DMEM medium supplemented with 10% FBS. After 4 days the medium was switched to a human ESC culture medium. Colonies activated the Oct4-GFP transgene after twelve days in culture (B–C) and are isolated for expansion after 17–18 days (D–E). After picking, single clones give rise to stable cell lines of FGF-iPSCs, which grow as round compacted colonies (F, H) expressing the Oct4-GFP transgene (G, I). Colonies expressing the Oct4-GFP (K, O, S) result positive for alkaline phosphatase activity (AP) (J), and SSEA-1 (M), Sox2 (P) and Nanog (T) immunoistochemistry. Merged figures for Sox2/Oct4-GFP and Nanog/Oct4- immunostainings are showed in Q and U, respectively. DAPI fluorescent staining is labeling cell nuclei (blue) in L, N, R.
Figure 2.
Derivation of murine iPS cells in the presence of bFGF by inducible lentiviral transduction.
(A) Schematic representation of the strategy used for the reprogramming of murine fibroblasts into iPS cells in the presence of LIF or bFGF. (B) Image of an EpiSC-like iPS cell colony derived in bFGF. (C) Schematic of the temporal analysis of the emergence of stable iPS colonies in the presence of bFGF. At day -1, murine embryonic fibroblasts are infected with the doxycycline-inducible reprogramming factors and rtTA. AT day 0, doxycycline is added to the culture media to initiate expression of the ectopic reprogramming factors. On day 1, the sample is split into dishes which are subsequently maintained in the presence of LIF or bFGF. Starting at day 5, doxycycline is withdrawn at set time intervals and emergence of stable, factor-independent colonies is analyzed at day 18. (D) Emergence of stable, factor independent colonies upon iPS reprogramming in the presence of bFGF (top panel) or LIF (bottom panel). Indicated on top is the day at which doxycycline was withdrawn from the culture to silence the ectopic reprogramming factors.
Figure 3.
The ES-like pluripotent state is dominant in the 129/BL6 genetic background.
(A) Comparative gene expression analysis among conventional murine ESCs, LIF-iPSCs, FGF-iPSCs and EpiSCs for candidate genes of pluripotency. Normalized expression intensity values (scaled median ratio) were obtained from Agilent whole-genome microarrays. Two biological replicates were used for all cell types. (B) Unbiased cluster analysis of global gene expression profiles of two independent FGF-derived iPS clones, two independent LIF-derived-iPS clones, two murine ES cell lines and two EpiSC cell lines. (C) qPCR quantification of the pluripotency associated genes Nanog, Sox2, Oct4, Rex1, Fbx15, Eras, Cripto, Cer1, Daz1, Fgf5, Sox17, Dkk1 and Lefty1 in LIF-iPSCs compared to FGF-iPSCs.
Figure 4.
FGF-iPSCs do not share crucial properties of EpiSCs.
(A) Colony morphology of FGF iPSCs (left), murine ES cells (middle) and EpiSCs (right). Note the dome-shaped, three dimensional morphology of the FGF-iPS cells. (B) Alkaline phosphatase (AP) staining of the cells in (A). FGF-iPSCs are expressing AP at levels comparable with murine ESCs. On the contrary EpiSCs are negative for this markers. (C) FISH analysis show that the majority of FGF-iPSCs contains two active X chromosomes. FGF-iPSCs robustly display X-inactivation upon differentiation. The percentage of FGF-iPSCs containing a Xist-cloud is quantified. (D) Approximately 90% of the undifferentiated FGF-iPSCs contain two active X-chromosomes, whereas 40% of the FGF-iPSCs display X-inactivation after 4 days of differentiation. (E) Double immunostaining for me3H3K27 and Oct4-GFP in two female FGF-iPS cell lines. Differentiated cells which lost Oct4 expression show an evident me3H3K27 positive nuclear staining corresponding to the inactivated X-chromosome (arrow). On contrary, undifferentiated Oct4-GFP+ cells do not present any me3H3K27 positive nuclear puncta. White arrow indicates the diagnostic staining for the inactive X chromosome. Inset in D is a higher magnification of a single cell nucleus with a me3H3K27 positive inactivated X-chr.
Figure 5.
FGF-iPSCs show activation of opposite TGFb and FGF intracellular pathways with respect to ESCs and LIF-iPSCs.
(A) Heat-map diagram of a small selection of self-renewal related genes that are in common between the three cell types. Shown are genes which are part of the pluripotency machinery, primitive-streak, Jak-Stat pathway and TGFb signaling pathway. (B) qPCR analysis for mRNA level quantification of the Nodal, Inhba, Bmp4, Gremlin-1 (Grem1) and Gdf3 genes in FSR-iPSCs and ESCs. (C) Immunoblotting showing the phosphorylation levels of the different SMAD and ERK1/2 proteins and compared among ESCs, LIF-iPSCs and FSR-iPSCs, tested as protein extracts of cell cultures grown in their normal conditions. In some conditions FGF-2 (bFGF) was withdrawn for six days. (D) General outline illustrating the state of activation of the two different molecular branches of the TGFb signaling and their key molecular components in FSR-iPSCs and LIF dependent stem cells (ESCs and LIF-iPSCs).
Figure 6.
Effect of Jaki, ALKi and LIF-antibody on FGF-iPSCs and LIF dependent ES and iPS cells.
(A) All cell samples were plated onto inactivated MEFs and treated for one week with Jaki, Alki or LIF-blocking antibody to evaluate the dependency from LIF/Stat pathway or TGFb pathway. The percentage of persisting Oct4-GFP positive colonies was evaluated at day 7. Reported values were normalized over the internal control (number of colonies in growth medium without inhibitors, black bars). The number of cell colonies are represented on the Y axis. After treatment with Jaki for 15 days the FGF-iPSCs maintein the morphology (B) and the Oct4-GFP expression (C). (D-E) AP staining reveal the mainteinance of positive colonies in treated cells. (F) After treatment the cells show no X chromosome inactivation. (G) Western blot show the expression of Stat3 and P-Stat3 in FGF-iPSCs and mESC. Stat3 phosphorylation is suppressed by Jaki. (H) Gene expression analysis by qPCR among mESCs, FGF-iPSCs, EpiSCs, FGF-iPSCs + Jaki and FGF-iPSCs + alphaLIF.
Figure 7.
Feeder-free culture of FGF-iPSCs.
The FGF-iPSCs growth in the absence of feeder layer as colonies expressing Oct4-GFP (A–B). (C–D) The colonies are positive for alkaline phophatase. (E–G) FGF-iPSCs maintain the expression of Nanog in a feeder free condition. (H) Staining for H3K27 on FGF-iPS colonies show the absence of X-chromosome inactivation.
Figure 8.
In vitro and in vivo pluripotency of FGF-iPSCs.
(A) FGF-iPSCs were detached with collagenase IV from the substrate to induce embryoid body (EB) formation. After 5 days in culture, the majority of EBs downregulate the GFP transgene. (B) EBs plated in N2-medium generate nestin positive neural progenitor cells as highlighted by immunofluorescence with its specific antibody. (C) EBs were plated on gelatin coated dishes containing DMEM medium supplemented with 20% FBS. After 20 days in such condition, islands of beating cardiomyocite became evident (see Movie S1). Cell differentiation was confirmed by immunoistochemistry for the mesodermal marker smooth muscle actin (Sma) (C) and for the endodermal marker Sox17 (D). (E–H) Ematoxylin and Eosin histological staining of teratomas, derived by subcutaneously injection of 1 million FGF-iPSCs, showed cartilage (E), adipose tissue (F), gut-like epithelium (G) and muscle (H). (I) Oct4-GFP positive FGF-iPSCs integrate into the inner cell mass of mouse blastocyst after morula aggregation and contribute, thereafter, to viable highly chimaeric animals (on the right) as compared to unmanipulated mice (on the left) (J). (K) Germiline contribution of FGF-iPS #5 and #9 chimera. (L) Genotyping demontrate the presence of the Oct4-GFP transgene in the offspring.