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. 2019 Apr 8;8:e42374. doi: 10.7554/eLife.42374

Figure 1. Induction of hypoxia in human and chimpanzee iPSC-CMs.

(A) Experimental design of the study. Cardiomyocytes were differentiated from iPSCs from eight human (H.sap), and seven chimpanzee (P.tro) individuals together with replicates from three individuals of each species. For the oxygen stress experiment iPSC-CMs in each species were cultured in normoxic conditions (10% O2 - condition A) for 6 hr prior to induction of hypoxia at 1% O2 for 6 hr (condition B). Following hypoxia, iPSC-CMs were re-oxygenated to 10% O2 for 6 hr (condition C), or 24 hr (condition D). (B) Peri-cellular O2 levels measured at each stage of the experiment for each experimental batch. Also see Figure 1—figure supplements 1–5.

Figure 1.

Figure 1—figure supplement 1. Cardiomyocytes can be differentiated from iPSCs from both humans and chimpanzees.

Figure 1—figure supplement 1.

(A) Schematic representation of the protocol used to differentiate cardiomyocytes from human and chimpanzee iPSC lines (see Materials and methods for a detailed description). (B) Immunostaining of cardiomyocyte-specific markers in iPSC-CMs from a representative human and chimpanzee individual. The cytoplasmic markers TNNT2 (green) and ACTN1 (red) are shown in the left panel. The nuclear marker IRX4 (green), together with TNNT2 is shown in the right panel. Nuclei are stained with Hoechst. Staining of iPSC-CMs incubated with a fluorescently-tagged secondary antibody, without a prior incubation with primary antibody, is included as a negative control in the bottom panel.

Figure 1—figure supplement 2. New human iPSC lines are pluripotent and display normal karyotypes.

Figure 1—figure supplement 2.

(A) Metaphase spreads of H22422 and H25237 iPSC lines. (B) Spontaneous differentiation of embryoid bodies to the three germ layers: ectoderm (expressing MAP2), endoderm (expressing AFP and HNF3B), and mesoderm (expressing SMA). (C) PCR products of the episomal vector used to reprogram the iPSCs from fibroblasts. While the H22422 line no longer has episomal reprogramming vector sequence present, the H25237 line tested positive for vector sequence.

Figure 1—figure supplement 3. iPSC-CM purity is similar in humans and chimpanzees.

Figure 1—figure supplement 3.

(A) Flow cytometry profiles of TNNT2-positive cells in (i) iPSC-CMs from a representative chimpanzee and human individual, (ii) iPSCs from a representative chimpanzee and human individual, (iii) iPSC-CMs incubated without TNNT2 antibody or viability stain (unlabeled), and (iv) iPSC-CMs incubated with viability stain only. Gates represent a conservative and lenient threshold for identifying TNNT2-positive cells. (B) The proportion of TNNT2-postive cells in each human and chimpanzee sample at both the lenient and conservative threshold.

Figure 1—figure supplement 4. Optimising the induction of a hypoxic response in iPSC-CMs.

Figure 1—figure supplement 4.

(A) Level of cellular toxicity (LDH activity) for two human iPSC-CM samples cultured at 21% oxygen, or at 10% oxygen for seven days. (B) mRNA expression of the BNP gene, a marker of cell stress, after iPSC-CMs are exposed to 6 or 24 hr of 1% oxygen, compared to iPSC-CMs cultured at 5% oxygen. mRNA levels were measured by qPCR, and expressed relative to 18S rRNA levels. Expression is measured in cells either cultured in media containing glucose or galactose as a carbohydrate source. (C) Level of cellular stress induced in the conditions described in (B).

Figure 1—figure supplement 5. Hypoxia induces oxidative damage in both species.

Figure 1—figure supplement 5.

(A) Levels of 8-OHdG, a measure of oxidative DNA damage, in the cell culture media from each individual. Values are normalized to normoxia or hypoxia values that is A (A-A), B (B-A), C (C-B), and D (D-B). (B) Levels of 8-iso-PGF2α, a measure of lipid peroxidation, in the cell culture media from each phase of the experiment in each species as in (A). (C) Raw values associated with 8-OHdG release. (D) Raw values associated with 8-iso-PGF2α release. Values from the baseline normoxic (brown), hypoxic (blue), short-term re-oxygenation (coral), and long-term re-oxygenation (dark red) conditions are shown in each species. Asterisk denotes a significant difference between conditions (*p<0.05, **p<0.005, ***p<0.0005).