Differential coupling of self-renewal signaling pathways in murine induced pluripotent stem cells

Abstract

The ability to reprogram somatic cells to induced pluripotent stem cells (iPSCs), exhibiting properties similar to those of embryonic stem cells (ESCs), has attracted much attention, with many studies focused on improving efficiency of derivation and unraveling the mechanisms of reprogramming. Despite this widespread interest, our knowledge of the molecular signaling pathways that are active in iPSCs and that play a role in controlling their fate have not been studied in detail. To address this shortfall, we have characterized the influence of different signals on the behavior of a model mouse iPSC line. We demonstrate significant responses of this iPSC line to the presence of serum, which leads to profoundly enhanced proliferation and, depending on the medium used, a reduction in the capacity of the iPSCs to self-renew. Surprisingly, this iPSC line was less sensitive to withdrawal of LIF compared to ESCs, exemplified by maintenance of expression of a Nanog-GFP reporter and enhanced self-renewal in the absence of LIF. While inhibition of phosphoinositide-3 kinase (PI3K) signaling decreased iPSC self-renewal, inhibition of Gsk-3 promoted it, even in the absence of LIF. High passages of this iPSC line displayed altered characteristics, including genetic instability and a reduced ability to self-renew. However, this second feature could be restored upon inhibition of Gsk-3. Collectively, our data suggest modulation of Gsk-3 activity plays a key role in the control of iPSC fate. We propose that more careful consideration should be given to characterization of the molecular pathways that control the fate of different iPSC lines, since perturbations from those observed in naïve pluripotent ESCs could render iPSCs and their derivatives susceptible to aberrant and potentially undesirable behaviors.

Figure 1. Culture conditions influence iPSC self-renewal and proliferation.

A ESCs or iPSCs (passage 21) were plated onto gelatin-coated dishes in KnockOut-DMEM (KO) supplemented with KO Serum Replacement (SR) and LIF in the presence or absence of 10%(v/v) Hyclone serum (HY) or GMEM supplemented with LIF and 10% (v/v) HY, as indicated. 48 hours after plating cultures were observed by light microscopy and photographs taken. Representative images are shown, scale bar = 200 µm. B The self-renewal capacity of ESCs and iPSCs were evaluated by alkaline phosphatase (AP) expression in clonal assays following 4 days culture in different conditions described in A. The average percentage of AP positive colonies ± SEM are shown from three independent experiments. Two-tailed paired t-tests indicate the following significance *** = p<0.005. C and D Cells where plated at day 0 in KO SR+LIF (C) or the same media supplemented with 10% (v/v) HY (D). Rapamycin (5 nM) or DMSO (1∶10000, as control) were added 24 hours after seeding, and kept in the medium for the duration of the experiment. Cells were harvested after 48 (D2) 72 (D3) and 96 (D4) hours and counted in triplicate. The average values ± SEM are shown from three independent experiments. E ESCs or iPSCs were seeded in KO SR+LIF at 8000 cells/cm². After 24 h DMSO 1∶10000 (ctrl), 5 nM Rapamycin (Rap), 10% (v/v) Hyclone serum (HY) or serum and rapamycin together (HY Rap) were added to the cultures. After 24 h treatment protein extracts were prepared, separated through 10% acrylamide gels using SDS PAGE and immunoblotted using the antibodies indicated.

Figure 2. iPSCs are less sensitive to LIF withdrawal than ESCs.

ESCs or iPSCs were plated at a concentration of 8000 cells/cm², harvested after 4 days and analyzed for Nanog-GFP expression by flow cytometry. Cells were grown in KO SR (A), KO SR supplemented with 10% (v/v) Hyclone serum (HY) (B) or GMEM supplemented with HY (C) in presence or absence of LIF, as indicated. Numbers shown on the histograms are the mean of FL1 fluorescence intensity (MFI). One representative experiment out of four is shown. D ESCs or iPSCs were seeded at 8500 cells/cm² in KO SR in the presence or absence of LIF. Protein extracts were prepared after 4 days incubation and immunoblotting performed with the antibodies indicated. Following detection of pY705 Stat-3, blots were stripped and reprobed to detect total levels of Stat-3. E and F Cells were seeded at 8500 cells/cm² in KO SR plus 10% (v/v) HY (E) or GMEM supplemented with 10% (v/v) HY (F), in the presence or absence of LIF. After 48 h protein extracts were prepared and immunoblotting performed as indicated.

Figure 3. Expression and phosphorylation status of key signaling intermediates in ESCs and iPSCs.

A and B ESCs or iPSCs were seeded 8000 cells/cm² in KO SR plus LIF. After 24 hours, 10% (v/v) Hyclone serum (HY) was added where indicated (A), or cells were washed 3 times with PBS before LIF-containing or LIF-free KO SR was added to the cultures as indicated (B). After a further 24 hours incubation, protein extracts were prepared, separated through 10% acrylamide gels using SDS-PAGE and immunoblotted using the antibodies indicated. Blots in A and B(i) were all generated using cell extracts from one replicate and those in B(ii) from an independent experimental replicate. C ESCs or iPSCs were seeded at 8500 cells/cm² in GMEM 10% (v/v) Hyclone serum (HY) plus LIF, then 24 h later washed and deprived of LIF and serum for 4 h. 5000 U/ml of LIF or 10% (v/v) HY were added and proteins extracted following 0, 5, 30 min treatment with LIF or 30 min treatment with serum. The phospho-proteins indicated were detected by immunoblotting of the same membrane, which had been cut referring to the size of the protein marker, then stripped and re-probed with the corresponding pan antibody. Results generated from the same blots are grouped, with each series terminating with the respective Gapdh as loading control.

Figure 4. iPSCs demonstrate enhanced responsiveness to Gsk-3 inhibition.

A ESCs and iPSCs were plated at a density of 8000cells/cm² in KO SR plus or minus LIF as indicated. After 24 hours, inhibitors were added to the medium at the following concentrations: LY294002: 5 µM; BIO: 2 µM; 1 m: 2 µM; DMSO 1∶10000 as control, and cultured for 4 days. Following treatment, Nanog-GFP expression was assessed by flow cytometry and the mean of fluorescence intensity (MFI) of Nanog-GFP was calculated for each condition. The histogram bars represent the variation in Nanog-GFP MFI in treated samples, compared to the KO SR plus LIF (DMSO treated) control condition, expressed as a percentage. The mean of three independent experiments ± SEM are shown. Two-tailed paired t-tests indicate the following significance values: *** = p<0.005, * = p<0.05 are referred to the DMSO plus LIF condition; ††† = p<0.005 † = p<0.01 are referred to the DMSO minus LIF condition; # = p<0.01 is referred to the plus LIF 1 m-treated condition. B The percentage of alkaline phosphatase positive ESC or iPSC colonies generated in KO SR plus LIF in the presence or absence of 10% (v/v) Hyclone serum (HY) were measured. The data represent the percentage variation in alkaline phosphatase (AP) positive colonies compared with the DMSO treated control. The average values ± SEM are shown from three independent experiments. Two-tailed paired t-test: *** = p<0.005, * = p<0.05 are referred to the DMSO plus LIF. C iPSCs were cultured in KO SR without LIF and supplemented with 10% (v/v) HY. The total numbers of colonies generated are shown and are compared with the total number of colonies generated by ESCs and iPSCs in KO SR plus LIF plus serum. The percentage of alkaline phosphatase positive colonies are shown in histogram D.

Figure 5. Effect of perturbation of key self-renewal signaling pathways in ESCs and iPSCs.

ESCs and iPSCs were seeded in KO SR plus or minus LIF as indicated. After 24 hours 2 µM 1 m and 10% (v/v) Hyclone serum (HY) alone or together (A), 2 µM 1 m (B), 5 µM LY294002 and 10% (v/v) HY (C) or DMSO 1∶10000 (in all controls), were added. After a further 24 hours proteins were extracted and immunoblotting performed as indicated. Blots were stripped and re-probed with Gapdh, pan Akt or Shp2 antibodies to assess loading.

Figure 6. High passage iPSCs exhibit altered morphology and response to stimuli.

Cells were plated at a density of 8500 cells/cm² in KO SR plus LIF. A iPSCs changed morphology after several passages (iii), compared with ESC (i) or iPSC low passage (ii), but reinstate an ESC-like morphology after 24 h treatment with 2 µM 1 m (iv). iPSCs were considered as low passage (iPSC LP) with passages <25 and high passage (iPSC HP) with passages >40. B Chromosome spreads of ESCs and low/high passage iPSCs. The arrow indicates chromosome aberrations in iPSC HP. One representative karyotype out of 20 from each cell line is shown. C 4 days after seeding, ESC or iPSC proteins were extracted, separated through 10% acrylamide gels using SDS-PAGE and immunoblotted using phospho-S15 p53 antibody. β-actin antibody was used as loading control. As a positive control, protein extracted from irradiated T lymphocytes was used. D Cells were plated at day 0 and then harvested after 48 (D2), 72 (D3) and 96 (D4) hours. Cells were counted in triplicate. The average doubling times ± SEM are shown from two independent experiments. Doubling times were calculated using free software available from www.doubling-time.com. Two-tailed paired t test: ** = p<0.01 * = p<0.05. E iPSCs low/high passage or ESCs were harvested 4 days after seeding and Nanog-GFP expression analyzed by flow cytometry. Contour plot graphs are shown and the missing Nanog-GFP low population in iPSC HP indicated by an arrow. F and G Alkaline phosphatase positive colonies were counted after growing in KO SR plus LIF supplemented with (F) or without 10% (v/v) Hyclone serum (HY, G). After 24 hours, 2 µM 1 m was added and kept in the medium until the cells were fixed and stained. The average values ±SEM are shown from three independent experiments.