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• W2890891427 abstract "•Blocking of PLK4-STIL module in hESCs/hiPSCs leads to:•Centrosome loss, prolonged and error-prone mitosis;•p53-dependent differentiation;•Reduction of pluripotency linked to altered protein turnover Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs. Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs. The centrosome, an organelle named by Theodor Boveri at the end of the 19th century, has been studied for a long time, but its functions and mechanisms of regulation are still incompletely understood. The centrosome typically acts as a microtubule organizing center (MTOC), taking part in cell division, cell shape organization, and cell motility (Conduit et al., 2015Conduit P.T. Wainman A. Raff J.W. Centrosome function and assembly in animal cells.Nat. Rev. Mol. Cell Biol. 2015; 16: 611-624Crossref PubMed Scopus (316) Google Scholar, Khodjakov and Rieder, 2001Khodjakov A. Rieder C.L. Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression.J. Cell Biol. 2001; 153: 237-242Crossref PubMed Scopus (287) Google Scholar, Piel et al., 2001Piel M. Nordberg J. Euteneuer U. Bornens M. Centrosome-dependent exit of cytokinesis in animal cells.Science. 2001; 291: 1550-1554Crossref PubMed Scopus (331) Google Scholar). Its core consists of two centrioles, microtubule-based structures with nine-fold radial symmetry, embedded in a protein matrix termed pericentriolar material (Bornens and Gönczy, 2014Bornens M. Gönczy P. Centrosomes back in the limelight.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014; 369https://doi.org/10.1098/rstb.2013.0452Crossref PubMed Scopus (25) Google Scholar, Nigg and Stearns, 2011Nigg E.A. Stearns T. The centrosome cycle: centriole biogenesis, duplication and inherent asymmetries.Nat. Cell Biol. 2011; 13: 1154-1160Crossref PubMed Scopus (420) Google Scholar). The centrosome duplicates once per cell cycle. As a cell divides, each daughter cell inherits one centrosome, so its number in the cells remains stable, similar to DNA content (Bornens and Gönczy, 2014Bornens M. Gönczy P. Centrosomes back in the limelight.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014; 369https://doi.org/10.1098/rstb.2013.0452Crossref PubMed Scopus (25) Google Scholar, Nigg and Stearns, 2011Nigg E.A. Stearns T. The centrosome cycle: centriole biogenesis, duplication and inherent asymmetries.Nat. Cell Biol. 2011; 13: 1154-1160Crossref PubMed Scopus (420) Google Scholar). To date, hundreds of centrosomal proteins participating in centrosome biogenesis have been identified (Andersen et al., 2003Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling.Nature. 2003; 426: 570-574Crossref PubMed Scopus (1051) Google Scholar, Gupta et al., 2015Gupta G.D. Coyaud É. Gonçalves J. Mojarad B.A. Liu Y. Wu Q. Gheiratmand L. Comartin D. Tkach J.M. Sally W.T. et al.A dynamic protein interaction landscape of the human centrosome-cilium interface.Cell. 2015; 163: 1484-1499Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), with PLK4-STIL module having a pivotal role in the orchestration of centriole duplication (Arquint and Nigg, 2016Arquint C. Nigg E.A. The PLK4-STIL-SAS-6 module at the core of centriole duplication.Biochem. Soc. Trans. 2016; 44: 1253-1263Crossref PubMed Scopus (87) Google Scholar, Bettencourt-Dias et al., 2005Bettencourt-Dias M. Rodrigues-Martins A. Carpenter L. Riparbelli M. Lehmann L. Gatt M.K. Carmo N. Balloux F. Callaini G. Glover D.M. SAK/PLK4 is required for centriole duplication and flagella development.Curr. Biol. 2005; 15: 2199-2207Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, Habedanck et al., 2005Habedanck R. Stierhof Y.-D. Wilkinson C.J. Nigg E.A. The Polo kinase Plk4 functions in centriole duplication.Nat. Cell Biol. 2005; 7: 1140-1146Crossref PubMed Scopus (612) Google Scholar, Tang et al., 2011Tang C.-J.C. Lin S.-Y. Hsu W.-B. Lin Y.-N. Wu C.-T. Lin Y.-C. Chang C.-W. Wu K.-S. Tang T.K. The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.EMBO J. 2011; 30: 4790-4804Crossref PubMed Scopus (182) Google Scholar). Overexpression of essential centrosome regulators, including PLK4, leads to centrosome amplification, whereas their depletion causes loss of centrosomes (Bazzi and Anderson, 2014Bazzi H. Anderson K.V. Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo.Proc. Natl. Acad. Sci. USA. 2014; 111: E1491-E1500Crossref PubMed Scopus (124) Google Scholar, Bettencourt-Dias et al., 2005Bettencourt-Dias M. Rodrigues-Martins A. Carpenter L. Riparbelli M. Lehmann L. Gatt M.K. Carmo N. Balloux F. Callaini G. Glover D.M. SAK/PLK4 is required for centriole duplication and flagella development.Curr. Biol. 2005; 15: 2199-2207Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, Habedanck et al., 2005Habedanck R. Stierhof Y.-D. Wilkinson C.J. Nigg E.A. The Polo kinase Plk4 functions in centriole duplication.Nat. Cell Biol. 2005; 7: 1140-1146Crossref PubMed Scopus (612) Google Scholar, Leidel et al., 2005Leidel S. Delattre M. Cerutti L. Baumer K. Gönczy P. SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells.Nat. Cell Biol. 2005; 7: 115-125Crossref PubMed Scopus (294) Google Scholar, Strnad et al., 2007Strnad P. Leidel S. Vinogradova T. Euteneuer U. Gönczy P. Regulated HsSAS-6 levels ensure formation of a single procentriole per centriole during the centrosome duplication cycle.Dev. Cell. 2007; 13: 203-213Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, Tang et al., 2011Tang C.-J.C. Lin S.-Y. Hsu W.-B. Lin Y.-N. Wu C.-T. Lin Y.-C. Chang C.-W. Wu K.-S. Tang T.K. The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation.EMBO J. 2011; 30: 4790-4804Crossref PubMed Scopus (182) Google Scholar). Deregulation of the centrosome duplication cycle is implicated in the etiology of various disorders such as ciliopathies, microcephaly, primordial dwarfism, and cancer (Chavali et al., 2014Chavali P.L. Pütz M. Gergely F. Small organelle, big responsibility: the role of centrosomes in development and disease.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014; 369https://doi.org/10.1098/rstb.2013.0468Crossref PubMed Scopus (107) Google Scholar, Gambarotto and Basto, 2016Gambarotto D. Basto R. Consequences of numerical centrosome defects in development and disease.in: Lüders J. The Microtubule Cytoskeleton: Organisation, Function and Role in Disease. Springer Vienna, 2016: 117-149Crossref Scopus (3) Google Scholar, Gönczy, 2015Gönczy P. Centrosomes and cancer: revisiting a long-standing relationship.Nat. Rev. Cancer. 2015; 15: 639-652Crossref PubMed Scopus (142) Google Scholar, Nigg et al., 2014Nigg E.A. Čajánek L. Arquint C. The centrosome duplication cycle in health and disease.FEBS Lett. 2014; 588: 2366-2372Crossref PubMed Scopus (65) Google Scholar). However, the consequences of centrosome abnormalities for cell fate have started to be revealed only recently. Inhibition of PLK4 depletes centrioles in various human somatic cell lines, leading to p53-dependent G1 arrest (Lambrus et al., 2015Lambrus B.G. Uetake Y. Clutario K.M. Daggubati V. Snyder M. Sluder G. Holland A.J. P53 protects against genome instability following centriole duplication failure.J. Cell Biol. 2015; 210: 63-77Crossref PubMed Scopus (93) Google Scholar, Wong et al., 2015Wong Y.L. Anzola J.V. Davis R.L. Yoon M. Motamedi A. Kroll A. Seo C.P. Hsia J.E. Kim S.K. Mitchell J.W. et al.Reversible centriole depletion with an inhibitor of Polo-like kinase 4.Science. 2015; 348: 1155-1160Crossref PubMed Scopus (264) Google Scholar). In contrast, in vivo study using Drosophila demonstrated that centrosomes are not required for a substantial part of fly embryogenesis (Basto et al., 2006Basto R. Lau J. Vinogradova T. Gardiol A. Woods C.G. Khodjakov A. Raff J.W. Flies without centrioles.Cell. 2006; 125: 1375-1386Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar). The requirement for correct embryo development has been further addressed in mice. Mouse embryos without centrosomes die during gestation (Bazzi and Anderson, 2014Bazzi H. Anderson K.V. Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo.Proc. Natl. Acad. Sci. USA. 2014; 111: E1491-E1500Crossref PubMed Scopus (124) Google Scholar, Hudson et al., 2001Hudson J.W. Kozarova A. Cheung P. Macmillan J.C. Swallow C.J. Cross J.C. Dennis J.W. Late mitotic failure in mice lacking Sak, a polo-like kinase.Curr. Biol. 2001; 11: 441-446Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Izraeli et al., 1999Izraeli S. Lowe L.A. Bertness V.L. Good D.J. Dorward D.W. Kirsch I.R. Kuehn M.R. The SIL gene is required for mouse embryonic axial development and left – right specification.Nature. 1999; 399: 691-694Crossref PubMed Scopus (170) Google Scholar), and amplification of centrosomes after PLK4 overexpression in developing mouse brain leads to microcephaly-like phenotype (Marthiens et al., 2013Marthiens V. Rujano M.A. Pennetier C. Tessier S. Paul-Gilloteaux P. Basto R. Centrosome amplification causes microcephaly.Nat. Cell Biol. 2013; 15: 731-740Crossref PubMed Scopus (173) Google Scholar). That being said, it is becoming clear that cellular outcomes of centrosome abnormalities differ between different models and perhaps even specific cell types (Basto et al., 2008Basto R. Brunk K. Vinadogrova T. Peel N. Franz A. Khodjakov A. Raff J.W. Centrosome amplification can initiate tumorigenesis in flies.Cell. 2008; 133: 1032-1042Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, Levine et al., 2017Levine M.S. Bakker B. Boeckx B. Moyett J. Lu J. Vitre B. Spierings D.C. Lansdorp P.M. Cleveland D.W. Lambrechts D. et al.Centrosome amplification is sufficient to promote spontaneous tumorigenesis in mammals.Dev. Cell. 2017; 40: 313-322Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, Marthiens et al., 2013Marthiens V. Rujano M.A. Pennetier C. Tessier S. Paul-Gilloteaux P. Basto R. Centrosome amplification causes microcephaly.Nat. Cell Biol. 2013; 15: 731-740Crossref PubMed Scopus (173) Google Scholar, Vitre et al., 2015Vitre B. Holland A.J. Kulukian A. Shoshani O. Hirai M. Wang Y. Maldonado M. Cho T. Boubaker J. Swing D.A. et al.Chronic centrosome amplification without tumorigenesis.Proc. Natl. Acad. Sci. USA. 2015; 112: E6321-E6330Crossref PubMed Scopus (52) Google Scholar). Human pluripotent stem cells (PSCs) encompassing both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are able to self-renew and to differentiate into all cell types in the human body (Takahashi et al., 2007Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14960) Google Scholar, Thomson et al., 1998Thomson J.A. Itskovitz-Eldor J. Shapiro S.S. Waknitz M.A. Swiergiel J.J. Marshall V.S. Jones J.M. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Crossref PubMed Scopus (12273) Google Scholar). Pluripotency, governed by a network of transcription factors including OCT-4, SOX-2, and NANOG (Jaenisch and Young, 2008Jaenisch R. Young R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming.Cell. 2008; 132: 567-582Abstract Full Text Full Text PDF PubMed Scopus (1115) Google Scholar, Kashyap et al., 2009Kashyap V. Rezende N.C. Scotland K.B. Shaffer S.M. Persson J.L. Gudas L.J. Mongan N.P. Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs.Stem Cells Dev. 2009; 18: 1093-1108Crossref PubMed Scopus (325) Google Scholar), is tightly connected to cell-cycle regulation (Becker et al., 2006Becker K.A. Ghule P.N. Therrien J.A. Lian J.B. Stein J.L. Van Wijnen A.J. Stein G.S. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase.J. Cell. Physiol. 2006; 209: 883-893Crossref PubMed Scopus (337) Google Scholar, Pauklin and Vallier, 2013Pauklin S. Vallier L. The cell cycle state of stem cells determines cell fate propensity.Cell. 2013; 155: 135-147Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Importantly, hESCs/hiPSCs hold great promise to model both physiological and pathophysiological aspects of human embryogenesis (Lancaster et al., 2013Lancaster M.A. Renner M. Martin C.-A. Wenzel D. Bicknell L.S. Hurles M.E. Homfray T. Penninger J.M. Jackson A. Knoblich J.A. Cerebral organoids model human brain development and microcephaly.Nature. 2013; 501: 373-379Crossref PubMed Scopus (2769) Google Scholar, Park et al., 2008Park I.-H. Arora N. Huo H. Maherali N. Ahfeldt T. Shimamura A. Lensch M.W. Cowan C. Hochedlinger K. Daley G.Q. Disease-specific induced pluripotent stem cells.Cell. 2008; 134: 877-886Abstract Full Text Full Text PDF PubMed Scopus (1801) Google Scholar, Shahbazi et al., 2016Shahbazi M.N. Jedrusik A. Vuoristo S. Recher G. Hupalowska A. Bolton V. Fogarty N.M.E. Campbell A. Devito L.G. Ilic D. et al.Self-organization of the human embryo in the absence of maternal tissues.Nat. Cell Biol. 2016; 18: 700-708Crossref PubMed Scopus (387) Google Scholar). Noteworthy, early passages of human PSCs seem prone to centrosome abnormalities (Brevini et al., 2009Brevini T.A.L. Pennarossa G. Antonini S. Paffoni A. Rebulla P. Scanziani E. De Eguileor M. Benvenisty N. Cell lines derived from human parthenogenetic embryos can display aberrant centriole distribution and altered expression levels of mitotic spindle check-point transcripts.Stem Cell Rev. Rep. 2009; 5: 340-352Crossref PubMed Scopus (37) Google Scholar, Holubcová et al., 2011Holubcová Z. Matula P. Sedláčková M. Vinarský V. Doležalová D. Bárta T. Dvořák P. Hampl A. Human embryonic stem cells suffer from centrosomal amplification.Stem Cells. 2011; 29: 46-56Crossref PubMed Scopus (40) Google Scholar). Given these unique properties, we elected to investigate the consequences of halted centrosome duplication cycle in early embryonic events using hESCs and hiPSCs. Here, we present our analyses of molecular and functional consequences of the inactivation of PLK4-STIL module and centrosome loss for human PSCs. We show that upon centrosome loss, the cells are in principle still able to undergo cell division. Such acentrosomal mitosis is twice as long and leads to mitotic errors and p53 stabilization, which is reflected by gradual loss of self-renewal potential. Interestingly, the observed p53 increase does not lead to significant apoptosis, but to loss of pluripotency and induction of differentiation. Finally, our data demonstrate that the loss of pluripotency regulators after PLK4 inhibition is p53-independent and linked to altered protein turnover. To assess the role of centrosomes in PSCs we used a PLK4 inhibitor, centrinone (Wong et al., 2015Wong Y.L. Anzola J.V. Davis R.L. Yoon M. Motamedi A. Kroll A. Seo C.P. Hsia J.E. Kim S.K. Mitchell J.W. et al.Reversible centriole depletion with an inhibitor of Polo-like kinase 4.Science. 2015; 348: 1155-1160Crossref PubMed Scopus (264) Google Scholar). First, we examined the efficacy of centrosome depletion in hESCs following treatment with centrinone. Using immunofluorescence staining for proximal centriolar marker Cep135 (Kleylein-Sohn et al., 2007Kleylein-Sohn J. Westendorf J. Le Clech M. Habedanck R. Stierhof Y.-D. Nigg E.A. Plk4-induced centriole biogenesis in human cells.Dev. Cell. 2007; 13: 190-202Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar) and distal centriolar marker CP110 (Chen et al., 2002Chen Z. Indjeian V.B. McManus M. Wang L. Dynlacht B.D. CP110, a cell cycle-dependent CDK substrate, regulates centrosome duplication in human cells.Dev. Cell. 2002; 3: 339-350Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar), we detected the loss of centrosomes in about 40% of hESCs after 2 days (Figures S1A and S1B), and after 3 days the centrosome was depleted in almost 85% of hESCs (Figures 1A and 1B ). We were also able to deplete centrosomes in hESCs using PLK4 or STIL short hairpin RNA (shRNA) (Figures S1C and S1D). It has been recently demonstrated that the loss of centrosomes is detrimental for proliferation of non-transformed human somatic cells, but has little effect on cancer cells (Fong et al., 2016Fong C.S. Mazo G. Das T. Goodman J. Kim M. O’Rourke B.P. Izquierdo D. Tsou M.F.B. 53BP1 and USP28 mediate p53-dependent cell cycle arrest in response to centrosome loss and prolonged mitosis.Elife. 2016; 5https://doi.org/10.7554/eLife.16270Crossref Scopus (107) Google Scholar, Lambrus et al., 2015Lambrus B.G. Uetake Y. Clutario K.M. Daggubati V. Snyder M. Sluder G. Holland A.J. P53 protects against genome instability following centriole duplication failure.J. Cell Biol. 2015; 210: 63-77Crossref PubMed Scopus (93) Google Scholar, Meitinger et al., 2016Meitinger F. Anzola J.V. Kaulich M. Richardson A. Stender J.D. Benner C. Glass C.K. Dowdy S.F. Desai A. Shiau A.K. et al.53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration.J. Cell Biol. 2016; 214: 155-166Crossref PubMed Scopus (123) Google Scholar, Mikule et al., 2007Mikule K. Delaval B. Kaldis P. Jurcyzk A. Hergert P. Doxsey S. Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest.Nat. Cell Biol. 2007; 9: 160-170Crossref PubMed Scopus (240) Google Scholar, Wong et al., 2015Wong Y.L. Anzola J.V. Davis R.L. Yoon M. Motamedi A. Kroll A. Seo C.P. Hsia J.E. Kim S.K. Mitchell J.W. et al.Reversible centriole depletion with an inhibitor of Polo-like kinase 4.Science. 2015; 348: 1155-1160Crossref PubMed Scopus (264) Google Scholar). Given reported similarities in cycle control between embryonic stem cells and cancer cells (Kim et al., 2010Kim J. Woo A.J. Chu J. Snow J.W. Fujiwara Y. Kim C.G. Cantor A.B. Orkin S.H. A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs.Cell. 2010; 143: 313-324Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar), we examined consequences of centrosome depletion for PSC proliferation. Intriguingly, centrinone-treated hESCs/hiPSCs showed impaired proliferation from day 2 and virtually halted their growth past day 5 (Figure 1C). In addition, we also observed a negative effect on proliferation of hESCs following STIL knockdown (Figure 1D). Noteworthy, the negative effect of centrosome loss on proliferation was even more pronounced in the case of hESC-derived neural stem cells (NSCs) (Figure S1E). On the other hand, centrinone treatment showed only a minor effect on proliferation of U2OS cells (Figure S1F), in agreement with the previous report (Wong et al., 2015Wong Y.L. Anzola J.V. Davis R.L. Yoon M. Motamedi A. Kroll A. Seo C.P. Hsia J.E. Kim S.K. Mitchell J.W. et al.Reversible centriole depletion with an inhibitor of Polo-like kinase 4.Science. 2015; 348: 1155-1160Crossref PubMed Scopus (264) Google Scholar), even though the efficiency of centrosome depletion was comparable with that of hESCs (Figure S1G). To corroborate this result, we examined the expression of Ki-67, a marker of proliferating cells. As shown in Figure 1E, centrinone treatment reduced expression levels of Ki-67. In addition, a decrease in the number of Ki-67+ cells was detected in the centrinone condition also by immunofluorescence (Figure S1H, quantified in Figure S1I). Centrosome loss has been reported to cause various mitotic defects in somatic cell lines (Sir et al., 2013Sir J.H. Pütz M. Daly O. Morrison C.G. Dunning M. Kilmartin J.V. Gergely F. Loss of centrioles causes chromosomal instability in vertebrate somatic cells.J. Cell Biol. 2013; 203: 747-756Crossref PubMed Scopus (87) Google Scholar, Wong et al., 2015Wong Y.L. Anzola J.V. Davis R.L. Yoon M. Motamedi A. Kroll A. Seo C.P. Hsia J.E. Kim S.K. Mitchell J.W. et al.Reversible centriole depletion with an inhibitor of Polo-like kinase 4.Science. 2015; 348: 1155-1160Crossref PubMed Scopus (264) Google Scholar). Indeed, we noted accumulation of rounded cells in the centrinone-treated cultures and following STIL knockdown (Figure 2A). Furthermore, our subsequent fluorescence-activated cell sorting (FACS) analysis proved that centrinone treatment leads to accumulation of hESCs/hiPSCs in G2/M phase (Figures 2B and 2C). Next, we analyzed the length of mitosis by live imaging of the reporter H2A-GFP line derived from the same paternal hESC line. As shown in Figure 2D, completion of mitosis between days 2 and 4 took for the treated cells approximately twice as long as controls. In addition, centrinone-treated hESCs showed 1.5-fold prolonged interphase on day 3 compared with control (Figure 2E). All these data indicated an intriguing possibility that centrosome-less hESCs are viable and able to divide, even though for a limited time for the latter. In agreement with this hypothesis we found bipolar mitotic spindles even in acentrosomal cells (Figure S2A). In addition, we quantified the number of cells successfully finishing mitosis in our live imaging experiments. We focused on mitoses past the third day of centrinone treatment, when the majority of treated cells already lacks centrosomes (Figures 1A and 1B). Interestingly, we found 68.1% ± 1.9% of cells able to successfully go through mitosis within the 30-hr period we examined. This observation suggested that acentrosomal mitoses seem possible, but also confirmed our earlier observation (Figure 1C) that proliferation after centrosome loss is inefficient (note 1.5 times longer interphase of centrinone-treated cells; Figure 2E). In addition, we observed cytokinesis failure in approximately 15% of divisions (Figure S2D). To fully prove that acentrosomal hESCs can divide, we performed live imaging experiments with γ-tubulin-GFP hESCs following centrinone treatment (Figure S2G). To conclude, these data argue that centrosome-depleted hESCs are in principle able to successfully finish mitotic division and give rise to two daughter cells, albeit only for a limited time. In the course of our experiments we noted that nuclei of centrinone-treated cells became bigger and acquired morphology different from control. In agreement with this, FACS analysis detected a modest increase of aneuploid cells after 3 days of centrinone treatment (Figures S2B and S2C). Since it is not possible to distinguish diploid cells residing in G2/M phase from tetraploid cells residing in G1 phase (Figures 2B and 2C) using this approach, it prompted us to quantify the chromosome number. The analysis was done at day 4, when the changes in cell morphology observed during live imaging were most pronounced. Previous work indicated that while centrosome loss during mouse embryogenesis does not lead to notable aneuploidy (Bazzi and Anderson, 2014Bazzi H. Anderson K.V. Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo.Proc. Natl. Acad. Sci. USA. 2014; 111: E1491-E1500Crossref PubMed Scopus (124) Google Scholar), somatic cell lines show an increase in chromosomal abnormalities after the centrosome loss (Sir et al., 2013Sir J.H. Pütz M. Daly O. Morrison C.G. Dunning M. Kilmartin J.V. Gergely F. Loss of centrioles causes chromosomal instability in vertebrate somatic cells.J. Cell Biol. 2013; 203: 747-756Crossref PubMed Scopus (87) Google Scholar, Wong et al., 2015Wong Y.L. Anzola J.V. Davis R.L. Yoon M. Motamedi A. Kroll A. Seo C.P. Hsia J.E. Kim S.K. Mitchell J.W. et al.Reversible centriole depletion with an inhibitor of Polo-like kinase 4.Science. 2015; 348: 1155-1160Crossref PubMed Scopus (264) Google Scholar). Interestingly, our analyses revealed that centrinone treatment of hESCs/hiPSCs led to changes in chromosome number (Figure 2F), arguing that centrosome loss promotes genome instability in PSCs. Next, to elucidate the survival potential of centrinone-treated cells, we assessed the number of early and late apoptotic cells by annexin V and propidium iodide (PI) staining. We found a modest difference in the number of viable (annexin V/PI negative) cells between centrinone condition and control (Figure 2G). Intriguingly, the proportion of apoptotic cells was notably elevated in hESC-derived NSCs following the centrinone treatment, in contrast to similarly treated cultures of hESCs/hiPSCs (Figure S2E). In addition, we compared the effects of centrinone with those of etoposide, a commonly used DNA-damage-inducing agent. Interestingly, while etoposide triggered a pronounced increase of apoptotic cells in hESC/hiPSC cultures, the percentage of apoptotic cells in centrinone-treated NSCs was similar to the NSC etoposide condition (Figure S2F). Key aspects of PSC biology, the ability to self-renew and to differentiate, are intimately connected to cell-cycle regulation (Becker et al., 2006Becker K.A. Ghule P.N. Therrien J.A. Lian J.B. Stein J.L. Van Wijnen A.J. Stein G.S. Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase.J. Cell. Physiol. 2006; 209: 883-893Crossref PubMed Scopus (337) Google Scholar, Pauklin and Vallier, 2013Pauklin S. Vallier L. The cell cycle state of stem cells determines cell fate propensity.Cell. 2013; 155: 135-147Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). Given the phenotypes we found, we examined the impact of centrosome depletion after blocking PLK4 or STIL on those two features. First, we observed that centrinone-treated cells lost typical stem cell morphology (Figure 3A), suggesting that centrosome loss affects stem cell differentiation. In agreement with this observation, we found a defect in polymerization of microtubules in centrosome-depleted hESCs (Figure S3A). Next, we examined expression of differentiation makers: ectodermal marker PAX-6, endodermal marker GATA-6, and mesodermal marker brachyury. Indeed, mRNA levels of all examined markers were upregulated after centrinone treatment (Figure 3B). Similar effects were confirmed also on the protein level (Figures 3C and S3B). Importantly, our analyses further revealed that protein levels of pluripotency markers OCT-4 and NANOG were decreased in centrinone-treated cells (Figures 3C and S3B). In addition, we detected higher protein levels of p53 in the centrinone conditions, thus confirming and extending previous observations on centrosome loss in somatic cells and mouse embryos (Bazzi and Anderson, 2014Bazzi H. Anderson K.V. Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo.Proc. Natl. Acad. Sci. USA. 2014; 111: E1491-E1500Crossref PubMed Scopus (124) Google Scholar, Insolera et al., 2014Insolera R. Bazzi H. Shao W. Anderson K.V. Shi S.-H. Cortical neurogenesis in the absence of centrioles.Nat. Neurosci. 2014; 17: 1528-1535Crossref PubMed Scopus (123) Google Scholar, Lambrus et al., 2015Lambrus B.G. Uetake Y. Clutario K.M. Daggubati V. Snyder M. Sluder G. Holland A.J. P53 protects against genome instability following centriole duplication failure.J. Cell Biol. 2015; 210: 63-77Crossref PubMed Scopus (93) Google Scholar, Mikule et al., 2007Mikule K. Delaval B. Kaldis P. Jurcyzk A. Hergert P. Doxsey S. Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest.Nat. Cell Biol. 2007; 9: 160-170Crossref PubMed Scopus (240) Google Scholar, Wong et al., 2015Wong Y.L. Anzola J.V. Davis R.L. Yoon M. Motamedi A. Kroll A. Seo C.P. Hsia J.E. Kim S.K. Mitchell J.W. et al.Reversible centriole depletion with an inhibitor of Polo-like kinase" @default.
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• W2890891427 date "2018-10-01" @default.
• W2890891427 modified "2023-10-17" @default.
• W2890891427 title "Inactivation of PLK4-STIL Module Prevents Self-Renewal and Triggers p53-Dependent Differentiation in Human Pluripotent Stem Cells" @default.
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• W2890891427 doi "https://doi.org/10.1016/j.stemcr.2018.08.008" @default.
• W2890891427 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/6178195" @default.
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