The survival advantage of culture-adapted hESCs under mitotic stress
Culture-adapted hESCs become highly resistant to various stressors such as DNA damage[11], single-cell dissociation, and YM155[8], a stemtoxic agent that selectively induces death in undifferentiated pluripotent stem cells[13] through SLC35F2[26,27]. In addition to the increased survival of hESCs under genotoxic stress, escape from cell death during mitosis has been suggested to increase aneuploidy during long-term culture[9]. Thus, we examined whether P4 hESCs exhibiting the survival trait under YM155 treatment (Fig. 1A) due to BCL-xL induction (encoded by BCL2L1, Fig. 1B) were more resistant to mitotic stress compared to P1 hESCs. P1 or P4 hESCs were treated with typical mitotic drugs targeting mitotic spindles such as nocodazole (Noc), spindle destabilizer, and taxol (Tax), as well as a spindle stabilizer, after which cell death was evaluated. Similar to YM155, P4 hESCs were highly resistant to mitotic stress inducers (Figs. 1C and D).
20q11.21 CNV was not sufficient to induce the survival trait in hESCs
Except for the role of BCL2L1 induction followed by CNV at locus 20q11.21 [10] or p53 dominant mutations [12], little is known regarding the mechanisms by which the survival trait is acquired during hESC culture adaptation. Further, no previous studies have determined how BCL2L1 transcription becomes upregulated, thus resulting in the survival advantage trait in culture-adapted hESCs. We observed that the survival trait became manifested in P3 (over 200 passages) and P4 (over 300 passages), but not P2 (over 100 passages) (Fig. S1A) with BCL2L1 expression comparable to that of P1 (up to 40 passage) hESCs [8], although CNV at locus 20q11.21 occurred in P2, P3, and P4 hESCs [8]. Similarly, hFmiPS1 (passage 20, normal iPSCs: hFmiPSC1-D1) and mutant cells (passage 30 with 20q11.21 CNV: hFmiPS2-DCB1) [28] underwent similar cell death by YM155 treatment (Fig. S1B) with comparable BCL2L1 expression [28]. These results imply that the acquisition of the survival trait via BCL2L1 induction may require additional events other than 20q11.21 CNV.
BCL2L1 induced survival associated with TPX2
As previously demonstrated, amplification of 20q11.21 was the most frequently occurring genome aberration and CNV of ID1, BCL21L, and HM13 at locus 2011q.21, suggesting that this was a marker of genome aberration [5]. However, intriguingly, not all genes at locus 20q11.21 were transcriptionally active in hESCs having 20q11.21 CNV (e.g., P2, P3, and P4 hESCs) (Fig. 2A). Among the genes in the 20q11.21 locus, TPX2, which was previously shown to induce aberrant mitosis in culture-adapted hESCs[17], was concurrently induced with BCL2L1 expression. Similarly, BCL-xL protein level was closely associated with TPX2 protein level in P3 and P4 hESCs where the survival trait was evident (Fig. 2C). Concretely, P4 hESCs with high TPX2 expression also expressed higher levels of BIRC5 (encoding Survivin), an anti-apoptotic factor highly expressed in undifferentiated hESCs [13,29] along with BCL2L1 (Fig. 2D). Considering the roles of TPX2 in cancer malignancy [30] and survival/chemoresistance [31], it is readily presumed that TPX2 expression may be associated with BCL2L1 induction and consequent survival traits. As predicted, depletion of TPX2 in P4 hESCs significantly attenuated BCL2L1 expression (Fig. 2E), suggesting that TPX2 induction somehow regulates BCL2L1 expression and confers the survival trait. The doxycycline (Dox) inducible TPX2 cell line established from P1 hESCs (iTPX2-hESCs) was used to explore this possibility (Fig. S2A). TPX2 mRNA (Fig. S2A) and protein (Fig. S2B) induction occurred in a dose-dependent manner. The signal from green fluorescence protein (GFP) conjugated with TPX2 was evident in the mitotic spindle where TPX2 is located during mitosis [32] (Fig. S2C). Using the iTPX2-hESCs, we examined whether BCL2L1 transcription was promoted by TPX2 induction with Dox. Surprisingly, TPX2 induction was sufficient to increase BCL2L1 mRNA (Fig. 2F) in a dose-dependent manner (Fig. 2G), as well as protein (Fig. 2H). It is important to note that iTPX2-hESCs were established from P1 hESCs with normal copy numbers and therefore the copy number of iTPX2-hESCs remained normal regardless of Dox treatment unlike P4 hESCs (Fig. 2I).
TPX2 induction rescues mitotic cell death of normal hESCs
We have previously demonstrated that TPX2, located at locus 20q11.21 along with BCL2L1, would be a putative driver for abnormal mitosis[17]. Using iTPX2-hESCs, cell death under mitotic stress (e.g., Noc) was determined after TPX2 induction by doxycycline (Dox). We noted that only a portion of cells expressed GFP-TPX2 even after Dox treatment for unknown reasons. Thus, a GFP negative population was used as an internal control and the cell death of the GFP positive population (expressing TPX2) of iTPX2-hESCs was monitored after mitotic stress (Fig. 3A). As predicted, GFP positive cells (expressing TPX2) were more resistant to mitotic stress induced by Noc (Fig. 3A) and Tax (Fig. S3A) than GFP negative cells. Similar results were obtained in a dose-dependent manner (Fig. 3B). The resistance to the mitotic stress by TPX2 induction was reproduced in other iTPX2-hESCs derived from hCHA3 (Fig. S3B). To rule out the potential pro-survival effect of GFP expression (rather than TPX2), P1 expressing enhanced green fluorescent protein (EGFP-P1) was co-cultured with P4 or iTPX2 hESCs (Fig. 3C), as basal TPX2 level in P4 and iTPX2 (due to leakage of Tet-O system [33]) was comparably higher than that of P1 hESCs (Fig. S3C). Consistently, the hESCs with high TPX2 expression (e.g., P4 and iTPX2) were more resistant to Noc-induced mitotic stress regardless of GFP expression (Figs. 3D and E). Higher TPX2 in P4 hESCs than P1 hESCs occurred constantly regardless of cell cycle phase[17]. To further assess whether high TPX2 expression was responsible for the resistance to mitotic stress, TPX2 was transiently depleted by siRNA#3 (Fig. S3D). Depletion of TPX2 in P4 hESCs at a similar level of TPX2 in P1 hESCs (Fig. S3D), sensitized the cells to Noc induced mitotic stress (Fig. 3F). It is also worth noting that stable knockdown or expression of TPX2 in hESCs was unsuccessful even after multiple attempts, suggesting that the basal level of TPX2 is critical for self-renewal of hESCs.
YAP/TEAD4 leads to BCL2L1 expression
Despite the occurrence of high mitochondrial priming to apoptosis[16], anti-apoptotic factors that are highly expressed in human undifferentiated ESCs such as BIRC5[13,29] maintain a fine balance between hESC life and death[20]. Particularly, BIRC5 or BCL2L1, anti-apoptotic factors highly expressed in P4 hESCs (Fig. 2D) that are downstream of YAP1[25], were upregulated in P4 hESCs. Considering the key roles of YAP1 in the survival of ESCs[23,24], we hypothesized that YAP1 activity may account for the acquisition of survival traits in culture-adapted hESCs. It has been previously demonstrated that Rho and Hippo regulation on YAP/TAZ activation is critical for the survival response of hESCs [24]. Similarly, geneset for Hippo signaling was lesser enriched in P3 and P4 hESCs (showing culture adapted phenotypes) based on geneset enrichment analysis (GSEA) than P1 and P2 hESCs (showing high mitochondrial cell death) (Figs. 4A and S4A). YAP1 activity determined by GTIIC reporters was significantly higher in P4 hESCs (Fig. 4B). Considering the pivotal role of YAP1 in the survival of both mouse and human ESCs [23,24], high YAP1 activity acquired during in vitro culture could further enhance the survival signal, thus resulting in the survival advantage. Although YAP1 mRNA level itself remained unaltered, BCL2L1 mRNA level was higher along with CTGF (encoded by CCN2 gene; i.e., a typical YAP1 downstream target [34,35]) in P4 hESCs than P1 hESCs (Fig. 4C). Despite similarities in YAP1 transcription, the YAP1 protein level was apparently stabilized with lower phosphorylated YAP1 (at S127) in P4 hESCs with higher TPX2 and TEAD4 expression (Fig. 4D). Consistently, nuclear YAP1 expression manifested along with TEAD4 protein expression in P4 hESCs (Fig. 4E). In contrast, depletion of YAP1 (Fig. 4F), TAZ (Fig. 4G), or TEAD4 (Fig. 4H) significantly decreased BCL2L1 expression along with CTGF in P4 hESCs.
Aurora-A stabilizes the YAP1 protein
Previously, Aurora A, a mitotic kinase strongly associated with TPX2 for spindle assembly, was shown to phosphorylate and stabilize YAP1 in cancer [9,36]. Constant activation of Aurora-A, which corresponded with TPX2 induction regardless of cell cycle phase, was a distinct cellular phenotype of P3 and P4 hESCs [17]. P4 hESCs with high Aurora-A (determined by phospho-Aurora A) and TPX2 activities exhibited high protein levels of BCL-xL and YAP1 (Fig. 5A). As transient depletion of TPX2 also significantly lowered the protein level of BCL-xL (encoded by BCL2L1) and YAP1, high TPX2 induction in P4 hESCs would be closely associated to YAP dependent BCL2-xL expression (Fig. 5B). Next, to confirm the increased protein level of YAP1 results from protein stability, YAP1 protein was monitored after cycloheximide (CHX) treatment to inhibit protein translation in P1 and P4 hESCs (Fig. 5C). Similarly, the increased protein level of YAP1 was evident in Dox-dependent TPX2 induction (Fig. 5D), whereas the YAP1 mRNA level remained unaffected (Fig. 5E). Aurora-A active phosphorylation was clearly induced by TPX2 induction in iTPX2-hESCs, which was concurrent with the increase of YAP1 protein expression (Fig. 5F). Similar to P4 hESCs, YAP1 protein and not TEAD4 (Fig. S4B) was highly stabilized after Dox treatment in iTPX2-hESCs (Fig. 5G). These data suggest that high YAP1 protein levels in culture-adapted hESCs result from high Aurora-A activity by TPX2 induction.
Inhibition of Aurora-A abrogated the resistance to mitotic stress by YAP1 destabilization
Given that the activity of Aurora-A in hESCs with high TPX2 expression appeared to stabilize YAP1, we next tested whether chemical inhibition of Aurora-A may destabilize YAP1 and sensitize hESCs with high TPX2 expression to mitotic stress. To this end, we first determined the concentration of Aurora-A inhibitor (MLN8237: MLN) for inhibition of Aurora-A in hESCs with high TPX2 expression (P4 and iTPX2-hESCs with Dox). Intriguingly, Aurora-A activity determed by its active phosphorylation was significantly reduced by 50nM of MLN treatment in P1 hESCs (Fig. S5A), where it remained active up to 100nM in P4 hESCs (Fig. S5B). Clear attenuation of active phosphorylation in P4 hESCs was distinct from 0.5mM of MLN treatment (Fig. 6A). Therefore, all downstream experiments for survival test in P4 were performed using 0.5mM of MLN. In pararllel with Aurora-A inhibition by MLN (Fig. 6A), BCL2L1 transcription was reduced along with CTGF and SERPINE1 [34,37] (Fig. 6B). Mitotic resistance in P4 hESCs, as determined in the sub G1 and 3N populations, was also significantly attenuated by additional MLN treatment with Noc (Fig. 6C). The same result was reproduced by flow cytometry to determine the live cell population (Fig. 6D). Consistently, marked stabilization of YAP1 by Dox in iTPX2-hESCs was reversed by MLN treatment (Fig. 6E). The reduced YAP1 protein by MLN resulted from the destabilization of YAP1 protein by MLN (Fig. 6F), which further led to BCL-xL protein downregulation (Fig. 6G). Accordingly, MLN treatment significantly sensitized GFP positive population (expressing TPX2) to mitotic stress in iTPX2-hESCs (Fig. 6H).