Activation of caspases 3 and 8 during the iPSC induction process
We were interested in caspase activities in the iPSC induction process because it had been known that some caspases play critical roles in cellular differentiation. For examples, caspases are known to be involved in the differentiation of hematopoietic stem cells (Kang et al., 2004
), osteoclasts (Szymczyk et al., 2006
), and embryonic stem cells (Dejosez et al., 2008
; Fujita et al., 2008
). We reasoned that because of their important roles in differentiation, caspases might also play some role in the reversal of the process: iPSC induction, which is essentially a de-differentiation process.
In order to monitor caspase activities in the iPSC induction process, we developed two non-invasive caspase reporters to monitor caspase 3 and 8 activities non-invasively. These two caspases were chosen because of previous reports of their involvement in differentiation (Dejosez et al., 2008
; Fujita et al., 2008
; Kang et al., 2004
; Szymczyk et al., 2006
). Our caspase reporters consist of a firefly luciferase-GFP fusion protein (LucGFP) linked to a polyubiquitin domain. In between the two moieties, a caspase cleavage site (either -IETD- for caspase 8 or –DEVD- for caspase 3) is inserted (, see supplementary Figure S1
for data validating the functionality and specificity of the reporters). The assumption is that when caspases are inactive, the reporter proteins will be recognized by proteasomes and degraded immediately because the polyubiquitin domain serves as tag for protein destruction by proteasomes. However, when caspase 3 or 8 is active, the polyubiquitin domain will be cleaved off the reporter protein, leading to enhanced GFP and luciferase signals because these reporter proteins are now not subject to direct proteasome recognition and degradation.
Caspases 3 and 8 are activated in the iPSC induction process
We transduced the caspase reporters into human IMR90 fibroblasts via recombinant lentivirus vectors. After puromycin selection to ensure stable integration of the reporters, we initiated the iPSC reprogramming process by infecting caspase reporter-transduced IMR90 cells with recombinant lentivirus vectors carrying 4 transcription factors (Oct-4, Sox2, Nanog, and Lin28, or OSNL in abbreviation) that are known to induce iPSC formation (Yu et al., 2007
). We chose to conduct the studies with the OSNL protocol to avoid the use of myc, which was part of the original Yamanaka OSKM protocol (Takahashi and Yamanaka, 2006
), because it was shown not to be essential for iPSC induction (Huangfu et al., 2008
; Kim et al., 2008
; Nakagawa et al., 2008
). In addition, it has known abilities to induce caspase activation (Evan et al., 1994
; Evan et al., 1992
; Hueber et al., 1997
), which might confound our results.
Interestingly, increased caspase 3 and 8 reporter activities were observed starting at day 3, continued to rise, and remained elevated during the rest of the observation period (). The specificity of the reporter was confirmed in supplementary Figure S1D
, which shows that expression of dominant negative caspase 3&8 genes in OSNL transduced IMR90 cells blocked caspase 3&8 reporter activation, respectively. In addition, these data show the dependency of caspase 3 activation on caspase 8 but not vice versa. Furthermore, our results indicated that reporter activation was not due to a decrease in proteasome function as a control reporter with no caspase cleavage site showed a small decrease in signals instead of an increase after OSNL transduction (supplementary Figure S1E
In iPSCs that eventually emerge from the OSNL-transduced cells, most (over 60%) of the colonies were positive for the caspase 3 reporter (supplementary Figure S2A
) when observed through GFP fluorescence(supplementary Figure S2B
).On the other hand, caspase 8 reporter activities were observed in less than 20% of iPSC colonies.
To confirm the above imaging results, western blot analyses of caspases 3 and 8 were carried out in IMR90 cells transduced with OSNL (). Our results indicated that while the levels of intact caspase 3 remained roughly the same in OSNL-transduced cells over time, there were progressive increases in activated caspase 3 with time (as represented by cleaved caspase 3 levels), indicating a steady increase in total caspase 3 levels. The level of activated caspase 3 remained robust in iPSCs. Interestingly, it was also quite high in H9 human embryonic stem cells (ESCs), similar to previous observations made in murine embryonic stem cells (Fujita et al., 2008
). There were significant increases in both total and activated caspase 8 levels in OSNL-transduced fibroblast cells (). The levels of both remained significant high during the entire course of observation. However, in contrast to caspase 3, there were lower amounts of total and activated caspase 8 in iPSCs that emerged from OSNL-transduced IMR90 cells (). Similarly, H9 human embryonic stem cells displayed low levels of total or activated caspase 8 (). We also carried out experiments to compare caspase 3 levels in iPSCs and iPSC-derived embryoid body (EB) cells because previously it was reported that caspase 3 levels increased during mouse ESC differentiation (Fujita et al., 2008
). Our results indicated that in EB cells derived from human iPSCs, total and activated caspase 3 levels held steady (Figure S2C
). In contrast, EB cells from mouse iPS cells had even higher levels of total and activated caspase 3 than mouse ESCs (Figure S2C
), consistent with previous observations (Fujita et al., 2008
Quantitative PCR analysis showe dincreased caspase 3 and 8 mRNA levels after OSNL transduction in IMR90 cells (supplementary Figure S2D
), consistent with western blot analysis results. In both IMR90-derived iPSCs and human H9 ESCs, caspase 3 mRNA levels are at about the same level as in IMR90 cells. However, caspase 8 mRNA levels in H9 human ESCs or iPSCs dropped precipitously to about 1/10 of the level in IMR90 cells (supplementary Figure S2E
), again consistent with western blot analysis.
Oct-4 expression alone is sufficient to induce caspase 3 and 8 activation
We next attempted to determine which of the four iPSC-inducing factors (e.g., OSNL) is responsible for caspase 3 and 8 activation. Our results () with caspase reporters indicate that Oct-4, which is consistently the most important transcription factor required in different iPSC induction protocols (Maherali and Hochedlinger, 2008
) and is able to induce iPSCs alone under certain circumstances (Kim et al., 2009a
; Kim et al., 2009b
), was responsible for caspase 3 and 8 activation. Among the other three factors, Nanog had a relatively small effect, and Sox 2 and Lin28 had minimal effects on caspase 3 and 8 activation (). Therefore, we conclude that Oct-4 is the factor primarily responsible for caspase 3 and 8 activation during OSNL-induced iPSC formation.
Oct-4 induced activation of caspases 3 and 8 and its effect on fibroblast cell viability
One important question is what kind of changes were induced by Oct-4 in the fibroblast cells. We observed significant morphological changes after Oct-4 transduction into IMR90 cells. These changes include smaller cell sizes, increased cellular densities, and faster growth rates (supplementary Figure S3A
, left panels). When these cells were stained for stem cell markers, robust Oct-4 expression was observed, as expected. However, only sporadic expression of other stem cell markers was observed (supplementary Figure S3A
, right panels). Despite clustered, colony type cellular growth, no iPS-like cells were observed over an extended period (10 weeks) of continuous culturing. These results indicated that forced Oct-4 expression induced significant morphological changes in IMR90 cells towards a more pluripotent state. However, its expression is not quite enough to push the cells into a full iPSC phenotype.
Western blot analysis confirmed that activities of both caspase 3 and caspase 8 were induced by Oct-4 transduction. The patterns of caspase 3 and 8 activation induced by Oct-4 paralleled those induced by OSNL (), thereby confirming the primary role of Oct-4 in mediating OSNL-induced caspase activation.
Additional western blot analysis confirmed induction of caspase 3 and 8 activation in IMR90 cells transduced with the original “Yamanaka Factors” (OSKM, or Oct-4+Sox2+Klf4+Myc) (Takahashi et al., 2007
) (Figure S3B
). A noteworthy difference is that OSKM appeared to induce caspase 3 and 8 activation at higher levels at day 10 (comparing , , and S3B
). Enhanced induction of caspase 3 and 8 activation is consistent with the extensive body of literature showing that myc, one of the four original Yamanaka factors, is able to induce caspase activation and cellular apoptosis (Evan et al., 1994
; Evan et al., 1992
; Hueber et al., 1997
It is not known whether observed caspase activation in OSNL- or OSKM-transduced cells causes apoptosis. Because myc is known for its ability to induce apoptosis in human fibroblast cells and was expressed at significantly higher levels in OSKM transduced cells than in OSNL transduced cells (Figure S3C
), we used the TUNEL assay to detect DNA fragmentation, which occurs at the end stage of apoptosis. Our results showed a significant amount of apoptosis (10–20% as determined by positive TUNEL signals) in OSKM-transduced IMR90 cells (). In comparison, only 1%-3% of OSNL-transduced cells were TUNEL positive (). This is consistent with previous findings that myc expression promotes fibroblast cells to undergo apoptosis (Evan et al., 1994
; Evan et al., 1992
; Hueber et al., 1997
Critical roles of caspase 3 and 8 activation in the iPSC induction process
In order to determine whether the observed caspase 3 and 8 activation had any functional relevance in iPSC induction, we attempted to down-regulate caspase activities in OSNL-transduced cells. In our initial experiment, we stably transduced CrmA (through lentiviral vectors), a cow pox virus protein known to suppress caspase 1 and 8 activities (Gagliardini et al., 1994
; Ray et al., 1992
), into IMR90 cells. After infecting these cells with an OSNL-encoding lentiviral vector cocktail, we found a significant (i.e., around 80%) suppression in the frequency of iPSC induction (). This was a very surprising result because logically one would expect that inhibiting caspase activation would lead to increased iPSC generation because of reduced cell death from stress induced by OSNL or OSKM transduction, which can induce apoptosis in transduced fibroblast cells ().
Caspases 3 and 8 are essential for iPSC induction
Because CrmA inhibits both caspase 1 and caspase 8, we subsequently used more specific approaches to pinpoint the exact caspase that was involved. First, we adopted an siRNA-based knockdown approach. To do this, we used lentiviral vectors encoding either caspase 1- or caspase 8-targeted shRNA to infect IMR90 cells. After selecting for cells with stable shRNA knockdown (as verified by western blot analysis), they were infected with the OSNL lentiviral vector cocktail. Our results indicate that expression of shRNA targeted to caspase 8 suppressed about 55% of iPSC formation (). In contrast, shRNA targeted to caspase 1 had no significant effect on iPSC induction frequency. These results indicated that the main inhibitory effect of CrmA on iPSC formation is mediated through caspase 8. Similarly, the importance of caspase 3,which can be activated by caspase 8, was demonstrated by targeted knockdown of its expression through shRNA. A significant reduction (around 50%) in iPSC induction was observed in caspase 3 knockdown IMR90 cells ().
The importance of caspase 8 was further demonstrated through transduction of the short version of the c-FLIP gene, which is a known cellular inhibitor of caspase 8 (Scaffidi et al., 1999
; Yeh et al., 2000
). Stable transduction of c-FLIP into IMR90 cells significantly attenuated the frequency of iPSC induction (about 50% reduction) (), again confirming the importance of caspase 8 in iPSC induction.
To provide definitive proof that the cleavage activities of caspases 3 and 8 are necessary for iPSC formation, we constructed lentiviral vectors encoding dominant-negative forms of caspases 3 and 8. The dominant negative form of the human caspase 8 gene (casp8-DN) encoding a protein virtually identical to the wild type except for a single amino acid change (i.e. C360A) (Stennicke and Salvesen, 1997
), which disables its proteolytic function, was cloned into a recombinant lentiviral vector. Similarly, the dominant negative form of caspase 3 (casp3-DN) (Stennicke and Salvesen, 1997
), which contains a single mutation (C163A) that disables its proteolytic function, was also cloned into a lentiviral vector. When IMR90 cells stably transduced with a lentiviral vector encoding casp8-DN were infected with the OSNL lentiviral cocktail, the iPSC induction process was completely shut down (, see Figure S4A
for an amplified photo of the alkaline phosphatase stained, iPSC-containing Petri dishes). No iPSC clones were observed after 25 days of culture, compared with around 550 iPSC clones/5×105
cells for wild type IMR90 cells. The dramatic inhibitory effect achieved through dominant negative caspase 8 expression indicated that the cleavage activity of caspase 8 was essential for iPSC induction.
Similar to caspase 8, we found that stable transduction of a dominant negative caspase 3 could significantly reduce the frequency of iPSC induction (about 80%, ). However, the induction process, despite being significantly attenuated by casp3-DN, was not completely shut down as in the case of casp 8-DN, indicating that there is some redundancy for the function of caspase 3, but not for that of caspase 8, in the iPSC derivation process.
The importance of caspase 8 was also confirmed in experiments conducted with the original Yamanaka protocol (Takahashi et al., 2007
; Yamanaka and Takahashi, 2006
). Transduction of casp 8-DN together with OSKM suppressed iPSC induction almost completely (over 95% suppression, see supplementary Figure S4B
), indicating the general importance of caspase 8 activation in different human iPSC induction protocols.
Experiments were also conduced to examine the importance of caspase inhibitors at various stages of iPSC induction. Our results (supplementary Figure S4C
) indicate that addition of caspase inhibitors can influence iPSC induction at all three stages (early, middle, and late) after OSNL transduction with early and late additions slightly more effective than middle stage addition.
A further experiment that supports positive roles for caspase 3 and 8 activation in iPSC formation came from following the fate of FACS sorted, caspase reporter-positive or -negative cells. IMR90 cells with caspase 3 or caspase 8 reporters () were transduced with OSNL and FACS sorted 3 days later for cells that were either positive (as determined by GFP expression) or negative for caspase 3 or caspase 8 reporter activities. It is estimated that around 20% of OSNL-transduced cells were positive for caspase 3 or caspase 8. Around 9×104 of those GFP+ or GFP- cells (, top panel) were then plated on feeder cells and observed for iPSC formation. We found that caspase reporter-positive cells were 6 times more likely to form iPSCs than caspase reporter-negative cells (, middle and lower panels), strongly suggesting a positive relationship between caspase activation and iPSC formation.
The effect of caspase 3 and 8 activation on iPSC formation in OSNL-transduced IMR-90 cells
One key question regarding the role of caspase 8 is whether the effects we observed were cell-autonomous or non-autonomous. It is theoretically possible that inhibition of caspase 8 attenuated some unknown, secreted paracrine factors that promote the formation of iPSCs from OSNL- or OSKM-transduced cells. To examine this possibility, we carried out experiments with IMR90 cells transduced with either casp8-DN or a control vector. The cells were mixed at the casp8-DN:control ratio of 9:1, and transduced with OSNL. The transduced cells were subsequently observed for iPSC colony formation. If the effects of caspase 8 were totally paracrine (or non-cell autonomous), we would expect that the ratio of iPSC colonies that originate from the two cell populations would equal to the ratio of the initial cellular mix, which is 9:1. If iPSC colonies were found mostly originate from control vector-transduced cells despite the fact they only accounted for 10% of the initial cell mix, this would indicate that the effect of casp8-DN expression is cell-autonomous. When iPSC colonies that emerged from the mixed cell population were selected, expanded, and analyzed through PCR, all 11 selected clones were proven to contain only the control vector sequences (). The probability for this to occur purely by chance is 10−11 (because the expected frequency for each of the 11 iPSC clones to originate from control vector transduced cells is 10−1 if the effect of caspase 8 suppression is non-cell autonomous) if we assume a non-cell autonomous mechanism. Therefore, our results strongly suggest that the effects of caspase 8 activation on iPSC induction are cell-autonomous.
Additional experiments also confirmed the importance of caspase activation in induction of iPSCs from mouse cells. In order to observe the activation of caspases 3&8 in murine iPSC induction process, we used a previously published secondary iPSC reprogramming system using mouse fibroblast cells derived from transgenic mice with doxycycline inducible OSKM genes already embedded within their genome (Stadtfeld et al., 2010
). These cells were transduced with our caspase reporters () and observed for caspase 3&8 induction. Our results indicate that both caspase 3&8 are strongly activated, as shown in . As a matter of fact, more than 50% of individual colonies from doxycycline induced cells showed strong activation of caspase 3&8 (top and middle panels) and many of these caspase activated colonies transformed into iPSC-like colonies after day 7. Western blot analysis confirmed the activation of both caspase 3&8 (, lower panel).
The functional importance of caspase activation was further demonstrated by use of mouse embryonic fibroblast cells derived from caspase 3 knockout mice. Our data (, top panel) indicate that caspase 3 deficiency in MEF cells significantly attenuated the number of iPSC colonies formed after OSKM transduction. Furthermore, even the few iPSC-like colonies that emerged from casp3−/− MEF cells were significantly smaller in size when compared with those from wild type cells. More importantly, they failed to expand when picked and plated with new feeder and ES medium. Not a single colony grew in three independent attempts (5 colonies were picked in each experiment). These data indicated that caspase 3 deficiency had a strong effect in preventing the full epigenetic reprogramming of MEF cells into iPS cells. In comparison, all iPSC-like colonies derived from wild type MEF cells expanded in secondary cultures successfully, indicating successful self renewal. In addition to strong AP staining (, middle panels), they stained positive for key ES markers (lower panels) and could form all three germ layers when injected into nude mice (, lower panels).
Further results supporting a role for caspases 3&8 come from experiments involving the use of chemical inhibitors of caspases (either general caspase inhibitors or specific caspase 3 or 8 inhibitors), which significantly inhibited iPSC formation from OSKM-transduced MEF cells (). In addition, the transduction of dominant negative caspase 3 or 8 genes significantly attenuated OSKM-induced iPSC formation from MEF cells (). These results suggest significant roles for caspases 3 and 8 in OSKM-mediated iPSC formation in murine fibroblast cells, consistent with our results from human iPSC induction experiments.
Retinoblastoma susceptibility protein is a key downstream target of caspases 3 and 8 in the iPSC induction process
We next tried to identify the mechanism through which caspase 8 facilitates iPSC induction. Because caspase 8 cleavage activities are clearly necessary in facilitating the iPS process, we reasoned that one or more of the cleavage substrates of caspases might be involved in the iPSC induction process. We further reasoned that caspase 8 may function through deactivation of factors involved in maintaining the differentiated state of fibroblasts. We eventually focused our attention on the retinoblastoma susceptibility (Rb) gene, which is a tumor suppressor gene that regulates cell cycle progression. It also has a key role in promoting and maintaining cellular differentiation (Dannenberg et al., 2000
; Jori et al., 2007
; White et al., 2005
). Previous studies have indicated that activated caspase 8 can cause cleavage and deactivation of the Rb protein through the activation of caspase 3 (An and Dou, 1996
; Dou et al., 1997
; Janicke et al., 1996
; Tan et al., 1997
). Inactivation of Rb through viral protein E1A (Ferrari et al., 2008
) or genetic deletion has been shown to facilitate cellular reprogramming to a stem cell-like state (Liu et al., 2009
We initiated our investigation into the role of the Rb protein by probing its status during the iPSC induction process. Western blot analyses () indicated that Rb protein was intact before the transduction of OSNL. However, a reduction in the full-length protein and an increase in cleaved Rb protein were obvious by day 3. By day 5 almost all full-length Rb protein was gone. Interestingly, starting at day 10, increases in both total and caspase-cleaved Rb fragments were observed. Furthermore, in iPSCs as well as in H9 hESCs, full-length Rb levels were similar to parental IMR90 fibroblast cells, indicating that Rb attenuation during iPSC induction was transient. The pattern of Rb cleavage during iPSC induction was almost identical to the patterns of caspase 3 and caspase 8 (). In particular, activated caspase 3 levels correlated very well with those of cleaved Rb, suggesting a very close relationship between the two. Rb proteins were mostly present at full-length in both iPSCs and ESCs at very robust levels, although weak, cleaved bands were clearly visible. Transduction of Oct-4 alone induced a similar Rb cleavage pattern (supplementary Figure S6A
), consistent with the primary role of Oct-4 in activating caspases 3 and 8. A further examination showed that Rb proteins are mostly phosphorylated in iPSCs (supplementary Figure S6B
), consistent with previous findings that Rb was inactive in ESCs (Burdon et al., 2002
; Savatier et al., 1994
Rb protein as a key downstream target of caspases 3 and 8 in the iPSC induction process
We next determined if Rb is functionally important for the iPSC induction process. We cloned the human Rb gene into a lentiviral expression vector and then stably transduced Rb into IMR90 cells. As shown in , over-expression of the Rb gene significantly reduced the frequency (about 50%) of OSNL-induced iPSC formation. The importance of caspase-mediated cleavage of Rb was further confirmed when Rb proteins with their caspase 3 cleavage sites mutated were evaluated for their ability to suppress iPSC formation in OSNL-transduced IMR90 cells (). Indeed we observed that caspase 3-resistant mutants could inhibit iPSC induction more effectively than wild type Rb. These results therefore clearly established that caspase-mediated Rb inactivation is an important step in iPSC induction.
The importance of deactivating Rb was further demonstrated through shRNA-mediated Rb knockdown. When we stably transduced an shRNA targeted to Rb into IMR90 cells, the frequency of OSNL-induced iPSC formation was almost doubled (), again consistent with Rb as an important barrier for iPSC induction.
Comparing Rb with p53 and p21 cell cycle regulating proteins in the iPSC induction process
In order to further examine the importance of caspase-mediated Rb cleavage in iPSC induction, we decided to examine the effect of human papillomavirus type 16 E7 (HPV16 E7) protein on the iPSC induction process. HPV16 E7 has been shown to effectively inhibit Rb function (Barbosa et al., 1990
; Dyson et al., 1989
; Gage et al., 1990
; White et al., 1994
). Co-transduction of E7 with OSNL increased the frequency of iPSC formation almost 5-fold (). Furthermore, E7 was able to partially alleviate the complete blockade of iPSC induction caused by casp8-DN (, top panel), strongly suggesting that caspase 8-mediated reprogramming proceeds at least partially through deactivating Rb. Consistent with these results, shRNA-mediated Rb knockdown was able to rescue the defect in iPSC induction partially in caspase deficient (casp3−/−) MEF cells mediated by OSKM (supplementary Figure S7A
Comparing Rb and other cell cycle regulating proteins
Western blot analyses were carried out to examine the status of Rb after OSNL and/or E7 transduction (, lower panel). OSNL or Oct-4 transduction caused significant attenuation of Rb levels. Inhibition of caspase 8 activity partially blocked OSNL transduction-mediated Rb attenuation. On the other hand, a dominant negative caspase 3 significantly blocked Rb cleavage after OSNL transduction. These data indicated that caspase 3 activity was required for Rb cleavage. Furthermore, rescue of iPSC formation in casp8-DN-transduced cells correlated with almost complete degradation of Rb in IMR90 cells transduced with OSNL+casp8-DN+E7, consistent with published literature suggesting the ability of E7 to destabilize Rb (Jones et al., 1997
Because recent reports indicated that the tumor suppressor gene p53 and its downstream gene p21 are involved in the iPSC formation process, we examined the status of these genes (). Our analysis did not reveal any caspase-mediated cleavage of p53 or p21 proteins. Furthermore, p53 protein levels did not significantly change during the course of iPSC induction or in iPSCs that eventually emerge. For p21, except a small transient increase at an early time (day 3), there was a small reduction of p21 protein levels at later times in OSNL-transduced IMR90 cells. In iPSCs and in H9 hESCs, p21 expression was completely suppressed, similar to previous reports (Hong et al., 2009
; Utikal et al., 2009
). In comparison, minimal changes in p53 and p21 levels were found in Oct-4-transduced IMR-90 cells (supplementary Figure S7B
A further experiment was done to examine the relative importance of Rb and p53 in the iPSC induction process. Previous studies have indicated that suppression of p53 could enhance the formation of iPSCs by 3–100 fold (Hong et al., 2009
; Kawamura et al., 2009
; Li et al., 2009
; Marion et al., 2009
; Utikal et al., 2009
). We carried out experiments to compare the effects of Rb down-regulation (through E7 expression or shRNA knockdown) versus p53 suppression in the iPSC induction process. Our results indicated a 2-fold increase in iPSC formation when a dominant negative p53 is co-expressed with OSNL, similar to Rb shRNA knockdown and lower than E7-mediated Rb suppression (), which caused a 5-fold increase in iPSC induction. When similar experiments were done in mouse MEF cells, the results were quite different: p53 knockdown mediated a 6-fold increase in iPSC formation while E7 transduction increased iPSC formation about 3-fold (supplementary Figure S7C
). The exact mechanism for the reversed roles of p53 and Rb in human and mouse fibroblasts is not clear. However, when western blot analyses of p53 and p21 were carried out in mouse MEF cells, we observed a clear induction of p53 and p21 that were further strengthened by HPV16 E7 transduction (supplementary Figure S7D
). Because previous studies have shown that Rb knockout results in activation of p19Arf, which can stimulate p53 and p21 expression through regulating MDM2 (Hsieh et al., 1999
; Pomerantz et al., 1998
; Zhang et al., 1998
), we examined p19Arf status. Our results indicate that p19Arf was not expressed during any stage of iPSC induction from passage 1 or 2 mouse MEF cells. This result was consistent with recent publications that report significant expression of p19Arf only occur in MEF cells after passage 3(Li et al., 2009
; Utikal et al., 2009
). In addition, p19Arf was not expressed in the mouse iPSCs that eventually emerged. Similarly, in human iPSC induction from IMR90 cells (<passage 10), there was also an absence of p14Arf expression (supplementary Figure S7E
) in cells transduced with OSNL or OSNL+shRb. Under those situations, knocking down Rb did not appear to promote p14Arf expression. Therefore, Rb and p53 pathways appear to play different roles in mouse and human iPSC induction, despite both being very important. The mechanisms for this difference are not clear at present.