We found, for the first time, that additional expression of DNp73 significantly increased human iPS cell generation by 12.6 folds, and also increased the kinetics, from 30 days to 21 days. These findings suggests that p73 is an important factor involved in the generation of human iPS cells. Furthermore, p73 has a unique dual mechanism, whereby DNp73 acts as an oncogene and increases iPS cell efficiency and kinetics, while TAp73 functions similarly to p53 in tumor suppression.
The strong impact of p53 on the iPS cell generation was discovered previously [3
] and Kawamura and group [4
] showed that cells deficient in the p53 gene expression simplified the iPS cell generation requirement with two factors: OCT4 and SOX2. In this study, we found that DNp73 had a similar effect of enhancement on iPS cell generation as p53. This system can also be applied to detect whether C-terminal splicing proteins of p73 or another p53 member, such as p63, are also important for iPS cell generation or cell differentiation.
Grob et al. [13
] found that DNp73 is capable of regulating the function of both TAp73 and p53 and is also strongly up-regulated by the TA isoforms and by p53, creating a feedback loop that tightly regulates the function of TAp73 and p53. In this study, our data strongly indicate that DNp73 expression decreases both p53 and TAp73, leading to an increase in Nanog gene expression, which subsequently benefits somatic cell reprogramming and enhances iPS cell differentiation resistance. The data also further suggests that DNp73 can be involved in tumorgenesis, at least teratoma-tumorgenesis. More extensive analysis of the expression pattern of DNp73 in other tumors and how the feedback loop is broken will require further studies.
Theunissen and Silva review [17
] on Nanog expression findings as follows: The loss of Nanog alleles are more prone to differentiate but do not lose pluripotency per se; Nanog is transiently required for the specification of the naive pluripotent epiblast and is also essential to finalize somatic cell reprogramming during induction of pluripotency; Nanog acts as a molecular switch to turn on the naive pluripotent programme in mammalian cells. However, Nanog expression also enforces tumor genesis in some recent researches. Most recently, a study by Grad I, et al. found that NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in tumour-inducing phenotype [18
]. Moon JH et al. [19
] reported that Nanog-induced dedifferentiation of p53-deficient mouse astrocytes into brain cancer stem-like cells. Those results underline the importance of a re-examination of the role of NANOG during reprogramming. Our results show that DNp73 derived iPS cells might increase Nanog expression. Consequently, the capacity of Nanog to resist differentiation can be regarded as a recapitulation of specification of somatic cell reprogramming, thus, resulting in a more teratomacarcinoma-like cell due to Nanog over expression. The results further indicate that p53 family members, including DNp73, might be key factors in reprogramming and cancer development. Therefore, our data provides a better understanding into tumour formation and cancer stem cell transformation, at least teratomatumorgenesis.
During iPS cell generation, global epigenetic modifications and chromatin changes, include histone modifications, DNA methylation, and chromatin remodeling [20
]. Patterson et al. [21
] found that genes normally unique to early embryos(LIN28A, LIN28B, DPPA4, and others)were not fully silenced in human iPS cell derivatives. LIN28 is an mRNA binding protein expressed in embryonic stem cells (Patterson), and is one of the key transcription factors for iPS cell generation [2
]. Our results showed that DNp73 might be involved in the generation and in vitro
differentiation of the iPS cells, suggesting, a link among epigenetic modifications, iPS cell gene expression and DNp73 in iPS cell generation and early embryonic development. However, the impact of DNp73 on epigenetic changes during iPS cell generation and derived cell lines has not been studied yet. A rapidly accumulating body of evidence suggests that there are important epigenetic differences between these two cell types, which seem to influence their tumorigenicity [22
]. Therefore further studies on these epigenetic modifications will be very interesting.
With small molecules, we developed an efficient set of human-induced pluripotent stem cell(iPSC) methods that improve iPSC generation by over 200-fold and cut iPSC formation time in half [23
]. Our results show TGF beta, MEK, WNT and ROCK signaling pathway inhibition or interference might strongly impact iPS cell generation efficiency and kinetics [24
]. Whether one or more of these pathways are shared with DNp73 needs further investigation.
Should iPS cells be applied to clinical application for effective treatment of human diseases? The most suspicious concerns are that iPS cells might be cancer prone. More and more data suggests that iPS cells are very similar to human tumor cells in the epigenetics as well as genetic mutation accumulated during the generation process. Hong et al. study found that some p53 inactivation in iPS cells produced tumors [3
]. Genetic safety is very critical for regenerative medicine. Further studies might be necessary to decipher whether the over expressed DNp73 is involved in the tumorigenesis, and how p73 functions in the normal iPS cells? It is not clear whether the p53 family members are a foe or friend in iPS cell generation and clinical application [23