Fig 1.
Cultivation and characterization of AD-iPSC colonies for pluripotency.
(a) AD-iPSCs one week after thawing on feeder cells. (b) AD-iPSCs two weeks in culture in high magnification. (c) Typical AD-iPSC morphology appears 4 weeks after thawing. (d-e) After two years freezing in liquid nitrogen the AD-iPSCs were positive for pluripotency associated alkaline phosphatase (AP) staining both on feeder cells and in a feeder-free system. (f) The AD-iPSCs were positive for embryonic stem cell markers such as NANOG. (g) Spontaneous neuronal differentiation of AD-iPSCs in vitro. (h-i) Typical AD-iPSCs morphology in low and high magnification.
Fig 2.
High basal spontaneous apoptosis in AD-iPSCs is caspase dependent.
(A) Pictures were taken 24 h after daily changing medium. (a-b) AD-iPSCs as a control with a lot of dead cells in the supernatant after 24 h in two different magnifications in contrast to iPSCs pretreated with 10 μm caspase inhibitor (c-d). (B) Employing FACS analysis the apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in AD-iPSCs pretreated with 10 μm caspase inhibitor (QVD-oph) for 2 h and 24 h. After pretreatment of AD-iPSCs with QVD-oph basal apoptotic cells decreased significantly compared to iPSCs without QVD-oph. (C) In support to our FACS analysis, apoptosis was quantified by determination of DNA fragmentation using ELISA cell death detection kit.
Fig 3.
Wortmannin induced apoptosis in iPSCs is caspase dependent.
(A) In support of our FACS analysis, apoptosis was quantified by determination of DNA fragmentation using ELISA cell death detection kit. The both iPSCs BIHi004-A and BIHi001-A were pretreated with QVD-oph or without QVD-oph and subsequently treated for additional 24h with 4μM wortmannin. Insets show picture of the ELISA with respect to color intensity. The color intensity is proportional to the level of apoptotic cells. (B) Pictures were taken 24 h after daily changing medium. (a,c) iPSCs as a control with a lot of dead cells in the supernatant after 24 h in two iPSCs in contrast to iPSCs pretreated with 10 μM caspase inhibitor (b,d).
Fig 4.
Strong apoptotic effect of wortmannin in iPSCs but not in parental fibroblasts and iPSC-derived neurons.
Pictures of fibroblasts, fibroblast derived iPSC lines and iPSC-derived neurons were taken after treatment with 4 μM wortmannin at different time points (1 h, 2 h, 4 h, 6 h, 24 h) of fibroblasts, fibroblast deriveded iPSCs and iPSC-derived neurons. In contrast to both fibroblasts (NFH-46) (A) and iPSC-derived neurons (C), iPSCs (B) showed clear sensitivity to wortmannin induced apoptosis, resulting in a massive cell death with increasing time. The magnifications of all images are 40x.
Fig 5.
Decrease of membrane potential and increase of ROS production upon wortmannin treatment in AD-iPSCs.
(A) Decreased membrane potential of mitochondria was determined by flow cytometry after TMRM+ staining in AD-iPSCs. Cells were treated with different concentrations of wortmannin (1 μM, 2 μM, 4 μM) for different times (1 h, 2 h, 24 h). Treated cells (open graphs) were compared to untreated controls (gray). (B) The quantitative data represent mean values of triplicate experiments +/-SD. (C) The production of ROS was determined after H2DCFDA staining in AD-iPSCs treated with two different concentrations of wortmannin (2–4 μM) or 4 μM of MK-2206 at two different time points (2h, 6h), by flow cytometry. Treated cells (open graphs) were compared to untreated controls (gray). (D) Quantitative data represent the median values of treated cells compared to control cells. ROS production was depicted as fold-increase, the control was set to 1. Two independent experiments with triplicates revealed comparable results
Fig 6.
Wortmannin significantly induced apoptosis in AD-iPSCs.
(A) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in iPSCs treated for 24 h with 10 μM QVD-oph, 10ng/ml TRAIL, 4 μM wortmannin (Wort.), 4 μM L-779,450, 4 μM BMS, and 50 nM MLN-8237 (AKA-I). Insets: Histogram examples of cells treated with wortmannin or BMS as compared to controls (Con.). Sub-G1 cell populations are indicated (sG1). (B) Whole cell cycle analysis after treatment with small molecules mentioned in A with respect to the amount of different phases (sub-G1, G1, S, G2/M) in percent. (C) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in orginal fibroblasts treated for 24 h with 10 μM QVD-oph, 10ng/ml TRAIL, 4 μM wortmannin, 4 μM L-779,450, 4 μM BMS, and 10 μM AKA-I. Insets: Histogram examples of cells treated with wortmannin or BMS compared to controls. Sub-G1 cell populations are indicated (sG1). (D) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in iPSC-derived neurons treated with increasing concentrations (0.5, 1, 2, 4 μM) of wortmannin for 24 h. Insets: Histogram examples of cells treated with two different concentrations of wortmannin (0.5/2 μM) compared to controls. (E) Measurement of apoptosis after treatment with 4 μM wortmannin for 1 h compared to DMSO. (F) Apoptosis (percentage of sub-G1 cells) by 10 μM QVD-oph, 4 μM wortmannin or the combination of both in iPSCs after 24 h. (G) Percentages of different cell phases of iPSCs treated with 10 μM QVD-oph, 4 μM wortmannin or the combination of both for 24 h. Means and SDs are shown of three independent experiments in triplicates. Statistical significance (*; p < 0.05) is indicated for comparison of control cells and wortmannin-treated cells.
Fig 7.
Wortmannin significantly induced apoptosis in BIHi001-A and BIHi004-A iPSC lines.
(A,E) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in iPSCs treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). (B,F) Whole cell cycle analysis after treatment with small molecules mentioned in A with respect to the amount of different phases (sub-G1, G1, S, G2/M) in percent. (C,D) Histogram examples of cells treated with BMS, AKA-I or wortmannin as compared to controls (DMSO). Sub-G1 cell populations are indicated (sG1). (G) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in orginal fibroblasts (HFF and NHDF) treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). Insets: Histogram examples of cells treated with AKA-I or wortmannin compared to controls (DMSO). Sub-G1 cell populations are indicated (sG1). (H,I) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in two iPSC-derived neurons treated for 24 h with 10ng/ml TRAIL, 4 μM L-779,450, 4 μM BMS, 50 nM MLN-8237 (AKA-I), 4 μM MK-2206, 4 μM wortmannin (Wort.). Insets: Histogram examples of cells treated with, BMS, AKA-I or wortmannin compared to controls (DMSO). Means and SDs are shown of three independent experiments in triplicates. Statistical significance (*; p < 0.05) is indicated for comparison of control cells and wortmannin-treated cells.
Fig 8.
Wortmannin induced apoptosis can be blocked by pancaspase inhibitor but not by p53 inhibitor.
(A,B) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in both BHIi001-A and BHIi004-A iPSC lines treated with 10 μM pancaspasen Inhibitor (QVD-oph, 2h pretreatment) and subsequently with 4 μM wortmannin (Wort.) for additional 24h. Insets: Histogram examples of cells treated with wortmannin, QVD or QVD combined with Wortmannin as compared to controls (Con.). Sub-G1 cell populations are indicated (sG1). (C-E) Apoptosis (percentage of sub-G1 cells) was determined by cell cycle analysis in AD-iPSCs, BIHi001-A and BIHi004-A, all three iPSC lines pretreated with 4μM, 10μM and 50μM p53 inhibitor (PFT-alpha) and subsequently with 1μM wortmannin (Wort.) for additional 24h. Insets: Histogram examples of cells treated with PFT-alpha alone or PFT-alpha combined with wortmannin compared to controls. Sub-G1 cell populations are indicated (sG1). Means and SDs are shown of three independent experiments in triplicates. Statistical significance (*; p < 0.05) is indicated for comparison of control cells and wortmannin-treated cells.
Fig 9.
Wortmannin induced apoptosis in iPSCs causes nuclear condensation and fragmentation detected by Hoechst-33258 staining.
(A) Phase contrast of untreated AD-iPSCs with sharp edges of round colonies (a) untreated AD-iPSC clones show mitotic cells, which can be seen by as intense blue (b-c). Phase contrast of wortmannin treated AD-iPSCs with frayed edges 1 h after treatment with 4μM wortmannin (d). A high proportion of cells showed clear indication of apoptosis by nuclear condensation and fragmentation, particularly pronounced at the edge of the colonies of AD-iPSCs (d-f) (examples indicated by arrows). B: Magnified images of untreated AD-iPSCs marked as mitotic cells (a), diffuse blue nucleus (b), sharp edge of untreated colony (c), magnified images of apoptotic cells with DNA condensation, DNA fragmentation (d-g). Changes in the morphology of the colonies one hour after treatment with wortmannin, and after 24 h complete detachment of the AD-iPSCs have been observed (h-i).
Fig 10.
Western blot analysis of key proteins of apoptosis in wortmannin treated AD-iPSCs.
Protein lysates were analyzed in AD-iPSCs after treatment with 4 μM wortmannin for different times. On the last lane of the blot at the right we subjected protein lysate of dead cells (DC) raised after 24 h in the supernatant to follow the basal apoptosis. Fourty μg of protein each were separated by SDS-PAGE (12%). Western blot analysis was used to monitor the expression of phosphorylated AKT (p-AKT, at serine 473) and total AKT, phosphorylated BAD and total BAD and p53. The anti-human Caspase-3 antibody used recognizes only the cleaved active form of caspase-3. The anti-human LC3A antibody recognizes both isomers, LC3I and LC3II. Furthermore we used anti-human BAX, BAK, BCL-2, BCL-XL and PUMA antibodies, against members of the Bcl-2 family. Beta-actin and Coomassie blue staining were used to confirm similar protein loading across samples.
Fig 11.
Comparative analysis of proteins between AD-iPSCs and AD-iPSC-derived neurons after wortmannin treatment.
Protein lysates of AD-iPSCs and iPSC-derived neurons (INC) were analyzed after treatment with 4μM wortmannin for different times. Fourty μg of protein each were separated by SDS-PAGE (12%). (A) Western blot analysis was used to monitor the expression of total and phosphorylated Akt (p-Akt, at serine 473). The anti-human LC3A recognizes both isoforms LC3I and LC3II and could also show phosphorylated LC3II. (B) iPSCs compared to iPSC-derived neurons and original fibroblasts. The anti-human Caspase-3 used recognizes only the cleaved active form of caspase-3. Beta-tubulin and Coomassie blue staining were used to confirm similar protein loading across samples.