From the simplest unicellular organisms to complex metazoans, cell polarity is a widespread characteristic that is essential for almost every aspect of biology. Proper polarization of cells is crucial for the establishment and maintenance of higher order structures such as tissue and organs. Cell polarity refers to the asymmetric distribution of various macromolecules and cellular structures, resulting in polarized architecture and function of the cell. Defects in cell polarity lead to various phenotypes, ranging from aberrant signaling, proliferation, cell adhesion and migration, cell fate determination and pluripotency, as well as embryonic lethality, neoplasia and cancer. Given the various roles for cell polarity in development and disease, the characterization of the components involved in polarity and their mechanisms of function is of great importance. My thesis work has encompassed three major projects, each of which is focused on understanding the role of cell polarity in development and disease. Although genetic screens in invertebrates have led to the identification of a number of cell-polarity proteins, similar systematic approach have not been undertaken in mammalian systems. The goal of my first project was to design and implement a high-throughput screen to systematically knockdown individual genes using siRNA, and then assess cell junction integrity as a measure of cell polarity. Given the importance of cell polarity to signaling pathways, I next sought to determine the mechanism by which cell polarity affects TGFβ and Hippo pathways, two important signaling pathways involved in development and disease. Lastly, by studying the acquisition of pluripotency by somatic cells, I uncovered a central role for cell polarity in the establishment and maintenance of pluripotency. Here I will present and discuss our discovery pertaining to the role of cell polarity in cell signaling and pluripotency.

From the simplest unicellular organisms to complex metazoans, cell polarity is a widespread characteristic that is essential for almost every aspect of biology. Proper polarization of cells is crucial for the establishment and maintenance of higher order structures such as tissue and organs. Cell polarity refers to the asymmetric distribution of various macromolecules and cellular structures, resulting in polarized architecture and function of the cell. Defects in cell polarity lead to various phenotypes, ranging from aberrant signaling, proliferation, cell adhesion and migration, cell fate determination and pluripotency, as well as embryonic lethality, neoplasia and cancer. Given the various roles for cell polarity in development and disease, the characterization of the components involved in polarity and their mechanisms of function is of great importance. My thesis work has encompassed three major projects, each of which is focused on understanding the role of cell polarity in development and disease. Although genetic screens in invertebrates have led to the identification of a number of cell-polarity proteins, similar systematic approach have not been undertaken in mammalian systems. The goal of my first project was to design and implement a high-throughput screen to systematically knockdown individual genes using siRNA, and then assess cell junction integrity as a measure of cell polarity. Given the importance of cell polarity to signaling pathways, I next sought to determine the mechanism by which cell polarity affects TGFβ and Hippo pathways, two important signaling pathways involved in development and disease. Lastly, by studying the acquisition of pluripotency by somatic cells, I uncovered a central role for cell polarity in the establishment and maintenance of pluripotency. Here I will present and discuss our discovery pertaining to the role of cell polarity in cell signaling and pluripotency.

Τα μηνιγγιώματα αποτελούν ιδιαίτερα συχνούς όγκους του Kεντρικού Νευρικού Συστήματος, όχι όμως επαρκώς μελετημένοι. Στην πλειοψηφία τους είναι καλοήθεις όγκοι (WHO grade I), όμως το 10% όλων των μηνιγγιωμάτων εμφανίζουν κακοήθη χαρακτηριστικά, όπως διήθηση των παρακείμενων ιστών, αυξημένα ποσοστά υποτροπής μετά από εξαίρεση, κ.α. Το σηματοδοτικό μονοπάτι Hippo είναι ένα διατηρημένο εξελικτικά μονοπάτι που εμπλέκεται σε διαδικασίες ρύθμισης του μεγέθους των οργάνων, κυτταρικού πολλαπλασιασμού, διαφοροποίησης και ανάπτυξης καρκίνου. Λίγα είναι γνωστά όσον αφορά το ρόλο του Hippo μονοπατιού στα μηνιγγιώματα. Σκοπός της παρούσας εργασίας είναι η μελέτη του Hippo μονοπατιού στα χαμηλής και υψηλής κακοήθειας μηνιγγιώματα. Χρησιμοποιήθηκαν 53 δείγματα από εξαιρεθέντα μηνιγγιώματα, 34 χαμηλής κακοήθειας και 19 υψηλής κακοήθειας, τα οποία μελετήθηκαν ανοσοϊστοχημικά ως προς την έκφραση των παραγόντων του Hippo CD44, YAP και TAZ. Παρατηρήσαμε στατιστικά σημαντική συσχέτιση μεταξύ της έκφρασης των CD44, ΥΑΡ και ΤΑΖ. Υπήρχε στατιστικά σημαντική συσχέτιση μεταξύ του βαθμού κακοήθειας και της έκφρασης των παραπάνω μορίων. Παρατηρήθηκε στατιστικά σημαντική διαφορά στην πυρηνική εντόπιση των ΥΑΡ και ΤΑΖ μεταξύ των υψηλής και χαμηλής κακοήθειας μηνιγγιωμάτων. Βάσει των παραπάνω αποτελεσμάτων, συμπεραίνουμε ότι το σηματοδοτικό μονοπάτι Hippo είναι απενεργοποιημένο στα υψηλής κακοήθειας μηνιγγιώματα με αποτέλεσμα οι μεταγραφικοί συνενεργοποιητές ΥΑΡ και ΤΑΖ να δρουν στον πυρήνα προάγοντας τον κυτταρικό πολλαπλασιασμό και διαδικασίες αντιαπόπτωσης. Αυτό, είναι ίσως εφικτό μέσω της δράσης του CD44 που αλληλεπιδρά με την μερλίνη, upstream ρυθμιστής του μονοπατιού. Απαιτούνται περαιτέρω μελέτες για τη διευκρίνηση του ακριβούς μηχανισμού. / Meningiomas are among the most common primary tumors of the Central Nervous System, but relatively understudied. The majority of meningiomas are benign tumors (WHO grade I), but almost 10% of all diagnosed meningiomas exhibit malignant features, such as invasiveness to the surrounding brain tissue, high recurrence rate, etc. Hippo signaling pathway is an evolutionary highly conserved protein kinase cascade involved in organ size control, cell proliferation and apoptosis, differentiation and cancer development. Very few are known about the role of Hippo pathway and meningiomas. In this study we attempt to find out whether Hippo path functions differently between high and low grade meningiomas. For this purpose paraffin embedded tissues obtained from 53 patients who underwent surgical removal of meningiomas were examined histoimmunologicaly. 34 out of 53 cases were low grade meningiomas and 19 out of 53 were high grade meningiomas. The paraffin sections were immunostained with CD44, YAP and TAZ antibodies, components of the Hippo signaling pathway. We observed statistically significant association between the expression of CD44, YAP and TAZ. There was a significant correlation between high grade meningiomas and expression of the examined factors. Moreover there was a significant difference in nuclear accumulation of YAP and TAZ between high and low grade meningiomas. Our findings suggest that in high grade meningiomas, Hippo pathway is inactivated and YAP and TAZ transcriptional co activators are able to insert nucleus and promote proliferation and antiapoptosis. This may be due to CD44 function, which interacts with merlin, an upstream regulator of the Hippo path. More experiments are required to verify the exact mechanism.

Το σηματοδοτικό μονοπάτι Hippo παίζει καθοριστικό ρόλο στην ομοιόσταση ιστών και οργάνων κατά την εμβρυογένεση και την ενήλικο ζωή ενώ όλο και περισσότερες μελέτες αναδεικνύουν τη σημασία του στην καρκινογένεση. Σκοπός της παρούσας μελέτης ήταν η διερεύνηση της έκφρασης των πρωτεϊνών YAP, TAZ, Ε-cadherin και b-catenin, στο Βασικοκυτταρικό καρκίνωμα του δέρματος (ΒΚΚ) και η συσχέτιση των ευρημάτων με την ενεργοποίηση της σηματοδοτικής οδού HIPPO, με την αλληλεπίδραση των σηματοδοτικών μονοπατιών Wnt/b-catenin και Hippo/YAP και με κλινικοπαθολογοανατομικές παραμέτρους του νεοπλάσματος. . Μέθοδοι και αποτελέσματα: Με την τεχνική της ανοσοϊστοχημείας ελεγχθηκε η έκφραση των πρωτεϊνών ΥΑΡ, ΤΑΖ, Ε-cadherin και b-catenin σε τομές παραφίνης μονιμομοποιημένου σε φορμόλη ιστού 95 περιστατικών ανθρώπινου ΒΚΚ. Η έκφραση και των δύο βασικών επιτελεστικών μορίων του Hippo (YAP,TAZ) βρέθηκε αυξημένη στον πυρήνα όλων των ιστολογικών τύπων ΒΚΚ και ήταν σημαντικά αυξημένη στους υψηλού κινδύνου υπότυπους. Τα μικροοζώδη, διαφοροποιούνταν τόσο από τα διηθητικά όσο και από τους χαμηλού κινδύνου υπότυπους εμφανίζοντας ενδιάμεση έκφραση για την ΥΑΡ όμως την υψηλότερη έκφραση για την ΤΑΖ μεταξύ όλων των ιστολογικών υποτύπων. Η E-cadherin στα περιστατικά μας διατηρούσε σε μεγάλο βαθμό τη μεμβρανική της έκφραση αν και μειωμένη σε σχέση με τη φυσιολογική επιδερμίδα, χωρίς σημαντικές διαφορές μεταξύ των ιστολογικών τύπων ή μεταξύ του όγκου συνολικά και της διηθητικής παρυφής. Τέλος, στα μισά περίπου περιστατικά μας διαπιστώθηκε πυρηνική έκφραση της β-catenin η οποία ήταν σημαντική στα διηθητικά σε σχέση με τα οζώδη, ενώ επίσης τα διηθητικά ΒΚΚ εμφάνιζαν τα υψηλότερα ποσοστά κυτταροπλασματικής έκφρασης της β-catenin μεταξύ όλων των ιστολογικών τύπων. Επίσης, η μεμβρανική έκφραση της β-catenin στο διηθητικό μέτωπο των υψηλού κινδύνου υποτύπων ήταν μειωμένη σε σχέση με τους χαμηλού κινδύνου. Συμπεράσματα: Το μονοπάτι Hippo φαίνεται ότι συμβάλλει στην παθογένεια του ΒΚΚ με έμφαση στους υψηλού κινδύνου υποτύπους, έχοντας κατά κύριο λόγο ογκογόνο δράση. Στην περίπτωση του ΒΚΚ, η Ε-cadherin δεν φαίνεται να αποτελεί ανωφερή ρυθμιστή του Hippo καθώς η διατήρηση της μεμβρανικής της έκφρασης δεν συνοδεύεται από σημαντικές αλλαγές στην υποκυτταρική εντόπιση της ΥΑΡ, γεγονός που ίσως αντανακλά τον ιστό προέλευσης του όγκου και οι νεοπλασματικές φωλεές διατηρούν αξιοσημείωτη κυτταρική συνοχή και επιθηλιακό φαινότυπο ακόμη και στην περίπτωση των επιθετικών υποτύπων. Η προφανής απαίτηση για Wnt/β-catenin σηματοδότηση στα ΒΚΚ θα πρέπει να ερευνηθεί περαιτέρω καθώς ενδέχεται να βρίσκεται υπό αρνητική ρύθμιση από παράγοντες όπως η ενεργοποίηση της non canonical Wnt σηματοδοτικής οδού που δρά ανταγωνιστικά ως προς την canonical Wnt σηματοδοτική οδό και η έκφραση σε υψηλά επίπεδα της E-cadherin στις μεμβράνες. Η παρατηρούμενη βιβλιογραφική ετερογένεια ως προς τη σημασία της Wnt σηματοδότησης στην παθογένεια του ΒΚΚ πιθανόν να αντανακλά βιολογική ετερογένεια ως προς την απαίτηση για Wnt σηματοδότηση εν γένει στο ΒΚΚ που πιθανόν αντικατοπτρίζει διαφορετική συμβολή της Wnt σηματοδοτικής οδού στα διάφορα στάδια εξέλιξης του όγκου ή/και στους διαφορετικούς ιστολογικούς υπότυπους. / Hippo pathway has emerged as a crucial component of organ and tissue homeostasis. Increasing number of studies highlight it’s significance in carcinogenesis. The aim of this study was to determine the expression of YAP, TAZ, Ε-cadherin and b-catenin in human Basal Cell Carcinoma (BCC) and correlate it with the activation status of Hippo signaling pathway, the interaction of Wnt/b-catenin και Hippo/YAP signaling pathways and clinicopathological features of the tumor. Materials and results: Paraffin-embedded tissue sections from 95 human BCC cases were processed by immunohistochemistry for the expression of YAP, TAZ, Ε-cadherin and b-catenin. Nuclear expression of both Hippo effector proteins (YAP,TAZ) was observed in 100% of cases and was strongly corellated with the high risk subtypes. Micronodular differed from both the invasive and low risk subtypes by showing intermediate nuclear YAP expression and the highest nuclear TAZ expression among all BCC subtypes tested. E-cadherin expression in our cases was largely membranous, though reduced compared to normal epidermis, with no significant differences between histologic subtypes or between the tumor overall and it’s invasive front. Finaly, nuclear β-catenin expression was observed in about half of our cases and was significantly increased in the infiltrative compared to the nodular subtype with the infiltrative BCC’s having the highest cytoplasmic β-catenin expression among all subtypes tested. Moreover, membranous β-catenin expression at the invasive front was significantly reduced in the high risk subtypes when compared to the low risk. Conclusions: We believe that Hippo signaling pathway holds a critical oncogenic role in the pathogenesis of BCC, expecially in the high risk subtypes. In the case of BCC, Ε-cadherin doesn’t appear to act as an upstream regulator of Hippo because it’s relatively stable membranous expression doesn’t seem to follow any variations in the subcellular localization of YAP, an observation probably reflecting the fact that skin is the tissue of origin for BCC. Neoplastic nests remain remarkably cohesive and retain an epithelial phenotype even in the high risk variants. The obvious demand for Wnt/β-catenin signaling in BCC development needs to be further invastigated because it seems likely being under negative regulation by factors like activation of the non canonical Wnt signaling cascade and increased expression levels of membranous E-cadherin. The observed variability in the literature regarding the significance of canonical Wnt signaling in BCC pathogenesis could be reflecting biological variability in the contribution of Wnt signaling in BCC pathogenesis overall, corresponding to differencies in Wnt signaling between the various stages of neoplastic development or/and the different histologic subtypes .

No description available.

According to the Centers for Disease Control, cancer is one of the leading causes of death in the United States. Characterized as a disease that develops as a result of an unstable genome, cancer is known to arise from numerous spontaneous mutations in the DNA of cells. Recent evidence shows that cancer cells within tumors are not self-reliant; rather, they progress along with other cells in their surrounding environment. Tumor cells recruit neighboring cells that, like the cancer cells, also become deregulated, forming the tumor stroma that aids in tumor progression. Within the stroma, cancer-associated fibroblasts (CAFs) play a vital role in the progression of cancer. Recent studies have found an important link between increased matrix stiffness surrounding the tumor and the invasion of the tumor. Thus, it is proposed that as the matrix stiffens, the tumor takes on more aggressive phenotypes. The transcriptional regulators YAP and TAZ (YAP/TAZ), key effectors of the Hippo pathway, are known to respond and influence matrix stiffening. In stiff matrix environments YAP/TAZ accumulate in the nucleus, and can drive transcriptional events. CAF's from late stage breast cancers have been found to exhibit increased YAP expression and increased ability to remodel and stiffen the extracellular matrix. Whether YAP or TAZ in these CAFs influences the metastatic properties of tumor cells is unclear. The present study aims to establish a link between YAP/TAZ activity in CAFs and cancer migration and invasion. We hypothesized that high nuclear activity of YAP/TAZ in fibroblasts would lead to non-autonomous signals that increase epithelial migration, and conversely that signals originating from epithelial cells affect YAP regulation in fibroblasts. We obtained CAFs from oral squamous cell carcinomas (OSCC) at various stages, and interestingly found that when CAFs obtained from stage III and stage IV tumors were co-cultured with OSCC cells they had the ability to cause OSCC cell migration. This CAF-induced migration was dependent on YAP/TAZ in the CAFs, as YAP/TAZ knockdown repressed this crosstalk. To gain insight into the mechanisms driving this process, transwell migration assays were conducted using NIH-3T3 fibroblasts engineered to overexpress YAP, or mutants of YAP, in doxycycline-inducible manner. We found that expression of YAP in NIH-3T3 cells, particularly a nuclear-localized YAP mutant, promoted the ability for OSCC cells to migrate in co-culture experiments. Media conditioned from these cells was sufficient to recapitulate this phenotype, suggesting that secreted factors from these fibroblasts may act as a signal that promotes migration. This activity of YAP was dependent on the ability for YAP to bind to the TEAD transcription factors, a major mediator of YAP transcriptional activity. Together these results indicate that nuclear YAP activity in fibroblasts can modulate the migration of neighboring cancer cells, suggesting that YAP plays a key role in stroma-cancer crosstalk during cancer progression.

Elucidating the molecular events that integrate the patterning, morphogenesis, and differentiation of epithelial progenitor cells into complex tissues is a primary focus of epithelial developmental biology research. Expansion and maintenance of epithelial progenitor populations is crucial for developmental events, but growth must be tightly coupled to consequent cellular differentiation and specialization. The Hippo pathway has surfaced as an important regulator of epithelial progenitor identity: nuclear activity of the Hippo effector Yap maintains epithelial progenitor status while Hippo-mediated nuclear exclusion of Yap by the Lats1/2 kinases induces differentiation. Extending this general theme into an additional organ system, the submandibular gland (SMG), as well as identifying upstream regulators of Yap and Lats1/2 in the developing lung was the goal of this work. Here, we describe important roles for Yap in the morphogenesis and patterning of lung and SMG epithelium, both of which are composed of highly organized branched structures. Epithelial-specific genetic ablation of Yap as well as its upstream negative regulators Lats1/2 was used to interrogate loss- and gain-of-function phenotypes, whereby Lats1/2 ablation is known to result in unrestricted nuclear Yap activity. Loss of Yap in the SMG resulted in a striking deficiency of Krt5/Krt14-positive epithelial progenitor populations accompanied by impaired branching morphogenesis. Deletion of Lats1/2 in the SMG resulted in a massive expansion of Krt5/Krt14-positive epithelial progenitor populations that failed to terminally differentiate. As epithelial progenitors in the lung and SMG begin to differentiate, they also acquire distinct morphologies. In both the lung and the SMG, Krt5-positive basal cells lie beneath a layer of Krt8/Krt19-positive luminal cells. We observed that luminal cells exhibited a columnar morphology while basal cells retained a cuboidal morphology, and that this difference correlated with the expression of the polarity protein Crb3. After ablating Crb3 in the developing lung epithelium, luminal cells were unable to polarize, exhibited aberrant nuclear Yap activity, and remained in a progenitor state. Crb3 functions to initiate Lats1/2 activity, promoting Yap phosphorylation and its consequent nuclear exclusion, which drives differentiation. Taken together, this work identifies essential roles for polarity/Hippo pathway-mediated control of Yap activity in epithelial progenitor expansion and differentiation. / 2018-07-09T00:00:00Z

Mitosis is a critically important and time sensitive cellular process that proceeds rapidly, typically completing in 15-45 minutes. Mechanisms have evolved to measure the duration of mitosis, resulting in the identification of aberrant cells that spend too long in mitosis. If non-transformed cells undergo a mitosis that exceeds 90 minutes, then the resulting daughter cells activate a durable G1 arrest and cease proliferating. The underlying mechanism acting to time the duration of mitosis is unknown. Here, we demonstrate that cells activate the Hippo pathway upon entry into mitosis, which initiates degradation of the pro-growth transcriptional co-activators YAP and TAZ. Consequently, prolonged mitosis leads to decreased YAP/TAZ levels in the following G1, thus enforcing cell cycle arrest. We reveal that inactivation of the Hippo pathway, which is common in solid tumors, is sufficient to restore YAP/TAZ levels following a prolonged mitosis, and cells born from this prolonged mitosis can progress through the cell cycle. We also demonstrate that Hippo pathway inactivation alters cell fate decisions in response to mitotic arrest. Antimitotics (e.g. Taxol) have long been used to permanently arrest cells in mitosis, which frequently results in mitotic cell death. It has long been recognized that some cancer cells are resistant to antimitotics; this resistance can arise from cells escaping mitosis into the G1 phase in a process termed mitotic slippage. The mechanisms underlying these cell fate decisions are poorly understood. Here, we demonstrate that inactivation of the Hippo pathway promotes mitotic slippage and overall survival in cells treated with antimitotics by increasing antiapoptotic protein expression. Our data suggest that inactivation of the Hippo pathway may promote resistance to antimitotic therapies by favoring the survival and proliferation of cells that have experienced a prolonged mitosis. Interestingly, we find that restoring Hippo signaling to cancer cells that are resistant to antimitotic therapies sensitizes them to antimitotics and promotes mitotic cell death. Overall, we illuminate a broad role for Hippo signaling in determining cell fate during mitosis and identify a novel mechanism by which resistance to antimitotic therapies can arise. / 2020-10-24T00:00:00Z

The Hippo signalling pathway is an evolutionarily conserved kinase cascade responsible for the cell proliferation, tissue growth and apoptosis during development and its dysregulation contributes to tumourigenesis. This signalling pathway was initially discovered in Drosophila and soon after that, it was shown to be highly conserved in mammals. The core Lats kinases of this tumour suppressive pathway phosphorylate and inhibit the downstream transcriptional co-activators YAP and TAZ, which are implicated in various cancers. Latest reports revealed various E3 ubiquitin ligases to negatively regulate the Hippo pathway through ubiquitination, yet few deubiquitinating enzymes have been described. In the present study, we report USP9X deubiquitinating enzyme as an essential regulator of the central components of this pathway. USP9X interacted strongly with Lats2 kinase and to a lesser extent with WW45, Kibra and Angiomotin family proteins. The knockdown of USP9X resulted in notable downregulation and destabilisation of Lats kinase and to lesser extents WW45, Kibra and Amot. This resulted in enhanced nuclear localisation of YAP and TAZ accompanied with activation of their target genes, CTGF and CYR61. USP9X was shown to stabilise Hippo components through its deubiquitinating activity. USP9X enzyme defective mutant lost the activity to stabilise Lats2, WW45, Kibra and Angiomotins through deubiquitination, leading to their ubiquitination. In the absence of USP9X, cells exhibited epithelial to mesenchymal transition phenotype and additionally gained anchorage-independent growth in soft agar. Moreover, USP9X knockdown disrupted acinar organisation of breast cells in three-dimensional acini cultures. In addition, YAP/TAZ target gene activation in USP9X knockdown cells could be rescued by knockdown of YAP, TAZ and TEAD2. Lastly, USP9X protein expression showed a positive correlation with Lats kinases, but negative correlation with YAP/TAZ in pancreatic cancer tissues as well as pancreatic and breast cancer cell lines. The results strongly indicate that USP9X cooperates with Lats2 and other important Hippo components to suppress tumour growth.

Cell based therapy using stem cell derived cardiomyocytes, has emerged as a potential therapeutic approach for cardiac diseases such as myocardial infarction and heart failure. Induced pluripotent stem cells (iPS cells) could be an ideal source of cardiomyocytes (iPS-CM). Challenges facing cell therapy include the high number of viable cells needed to survive in pathological conditions. The Hippo signalling pathway has been described as a key pathway involved in regulating cardiomyocyte proliferation and survival in both embryonic and adult hearts. We hypothesise that modification of the Hippo pathway will enhance the efficiency of iPS-CM generation and will increase iPS-CM survival and viability in pathological conditions. Skin fibroblasts were reprogrammed to iPS cells and then differentiated to cardiomyocytes. The Hippo signalling pathway was modified by genetic ablation of MST1, a major upstream regulator of the Hippo pathway, or by overexpressing YAP, the main downstream effector of the pathway. Cell proliferation was analysed using an EdU incorporation assay and staining for cytokinesis markers Ki67 and phospho-histone H3. Cell death and viability were analysed by measuring caspase 3/7 and MTT activity and by trypan blue staining in both normal and hypoxic conditions (CoCl2 treatment). Analysis of cell proliferation shows that genetic ablation of Mst1 leads to significantly increased proliferation (+12±1.5% P < 0.001), survival and viability (+20±4.3% P < 0.001) of iPS cells in both normal and hypoxic (CoCl2 treatment) conditions compared to controls. In addition, overexpression of YAP, which is normally inhibited by upstream Hippo pathway components, and overexpression of mutated constitutively active form of YAP (S127A) increases cell proliferation in iPS-CM compared to control iPS-CM as shown with EdU assay (46±2.60% P < 0.01) and Ki67 staining (4.9±0.9% P < 0.001). Overexpression of YAP leads to up regulation of genes associated with inhibition of apoptosis and promotion of cell proliferation. Preliminary studies show mouse iPS-CM are retained in the myocardium following intra-cardiac injection and do not cause any adverse effects confirmed with histological, echocardiography and electrocardiogram analysis. In conclusion targeting the Hippo pathway in iPS cells and iPS-CM significantly increases proliferation and survival in both normal and hypoxic conditions. Therefore, modulation of the Hippo pathway could become a new strategy to enhance the therapeutic potential of iPS-CM.