https://hdl.handle.net/10481/31032 2026-04-12T04:11:37Z https://hdl.handle.net/10481/111612 A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification Bueno, Clara; Montes, Rosa; Melen, Gustavo J; Ramos Mejía, Verónica; Real Luna, Pedro José; Ayllón, Verónica; Sanchez, Laura; Ligero, Gertrudis; Gutierrez-Aranda, Iván; Fernández, Agustín F; Fraga, Mario F; Moreno-Gimeno, Inmaculada; Burks, Deborah; Plaza-Calonge, María del Carmen; Rodríguez-Manzaneque, Juan Carlos; Menéndez, Pablo The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in infants. Although it is well established that MLL-AF4 arises prenatally during human development, its effects on hematopoietic development in utero remain unexplored. We have created a human-specific cellular system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs). Functional studies, clonal analysis and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic, functional and gene expression impact. MLL-AF4 acts as a global transcriptional activator and a positive regulator of homeobox gene expression in hESCs. Functionally, MLL-AF4 enhances the specification of hemogenic precursors from hESCs but strongly impairs further hematopoietic commitment in favor of an endothelial cell fate. MLL-AF4 hESCs are transcriptionally primed to differentiate towards hemogenic precursors prone to endothelial maturation, as reflected by the marked upregulation of master genes associated to vascular-endothelial functions and early hematopoiesis. Furthermore, we report that MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells. This work illustrates how hESCs may provide unique insights into human development and further our understanding of how leukemic fusion genes, known to arise prenatally, regulate human embryonic hematopoietic specification. https://hdl.handle.net/10481/111610 Identification of Cdca7 as a novel Notch transcriptional target involved in hematopoietic stem cell emergence Guiu, Jordi; Bergen, Dylan J. M.; De Pater, Emma; Islam, Abul B. M. M. K.; Ayllón Cases, Verónica Pilar; Leonor, Gama-Norton; Ruiz-Herguido, Cristina; González, Jessica; López-Bigas, Nuria; Menéndez, Pablo; Dzierzak, Elaine; Espinosa, Lluis; Bigas, Anna Hematopoietic stem cell (HSC) specification occurs in the embryonic aorta and requires Notch activation; however, most of the Notch-regulated elements controlling de novo HSC generation are still unknown. Here, we identify putative direct Notch targets in the aorta-gonad-mesonephros (AGM) embryonic tissue by chromatin precipitation using antibodies against the Notch partner RBPj. By ChIP-on-chip analysis of the precipitated DNA, we identified 701 promoter regions that were candidates to be regulated by Notch in the AGM. One of the most enriched regions corresponded to the Cdca7 gene, which was subsequently confirmed to recruit the RBPj factor but also Notch1 in AGM cells. We found that during embryonic hematopoietic development, expression of Cdca7 is restricted to the hematopoietic clusters of the aorta, and it is strongly up-regulated in the hemogenic population during human embryonic stem cell hematopoietic differentiation in a Notch-dependent manner. Down-regulation of Cdca7 mRNA in cultured AGM cells significantly induces hematopoietic differentiation and loss of the progenitor population. Finally, using loss-of-function experiments in zebrafish, we demonstrate that CDCA7 contributes to HSC emergence in vivo during embryonic development. Thus, our study identifies Cdca7 as an evolutionary conserved Notch target involved in HSC emergence. J. Guiu was a recipient of Formación Personal Investigador (FPI) BES-2008-005708. This research was funded by grants from the Ministerio de Economía y Competitividad, FEDER (SAF2007-60080, PLE2009-0111, SAF2010-15450, and SAF2013-40922R), Red Temática de Investigación Cooperativa en Cáncer (RTICC; RD06/0020/0098 and RD12/0036/0054), Association for International Cancer Research (AICR; 13-0064), Agència de Gestió d’Ajuds Universitaris i de Recerca (AGAUR; 2009SGR-23, CONES2010-0006) to A. Bigas, the Spanish Association against Cancer Research (AECC) to A. Bigas and P. Menendez, Fondo de Investigación Sanitaria (FIS; PI10/00449) to P. Menendez, and ZonMW TOP (40-00812-98-11068) to E. Dzierzak and E. De Pater. https://hdl.handle.net/10481/111607 The Notch ligand DLL4 specifically marks human hematoendothelial progenitors and regulates their hematopoietic fate Ayllon, Veronica; Bueno, Clara; Ramos Mejía, Verónica; Navarro-Montero, Oscar; Prieto, Cristina; Real Luna, Pedro José; Romero, Tamara; García-León, María José; Toribio, Maria Luisa; Bigas, Anna; Menendez, Pablo Notch signaling is essential for definitive hematopoiesis, but its role in human embryonic hematopoiesis is largely unknown. We show that in hESCs the expression of the Notch ligand DLL4 is induced during hematopoietic differentiation. We found that DLL4 is only expressed in a sub-population of bipotent hematoendothelial progenitors (HEPs) and segregates their hematopoietic versus endothelial potential. We demonstrate at the clonal level and through transcriptome analyses that DLL4(high) HEPs are enriched in endothelial potential, whereas DLL4(low/-) HEPs are committed to the hematopoietic lineage, albeit both populations still contain bipotent cells. Moreover, DLL4 stimulation enhances hematopoietic differentiation of HEPs and increases the amount of clonogenic hematopoietic progenitors. Confocal microscopy analysis of whole differentiating embryoid bodies revealed that DLL4(high) HEPs are located close to DLL4(low/-) HEPs, and at the base of clusters of CD45+ cells, resembling intra-aortic hematopoietic clusters found in mouse embryos. We propose a model for human embryonic hematopoiesis in which DLL4(low/-) cells within hemogenic endothelium receive Notch-activating signals from DLL4(high) cells, resulting in an endothelial-to-hematopoietic transition and their differentiation into CD45+ hematopoietic cells. https://hdl.handle.net/10481/111588 FLT3 activation cooperates with MLL-AF4 fusion protein to abrogate the hematopoietic specification of human ESCs Bueno, Clara; Ayllon, Veronica; Montes Lorenzo, Rosa María; Navarro-Montero, Oscar; Ramos Mejía, Verónica; Real Luna, Pedro José; Romero-Moya, Damia; Araúzo-Bravo, Marcos J; Menendez, Pablo Mixed-lineage leukemia (MLL)-AF4 fusion arises prenatally in high-risk infant acute pro-B-lymphoblastic leukemia (pro-B-ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hematoendothelial specification but was insufficient for transformation, suggesting that additional oncogenic insults seem required for MLL-AF4-mediated transformation. MLL-AF4+ pro-B-ALL expresses enormous levels of FLT3, occasionally because of activating mutations, thus representing a candidate cooperating event in MLL-AF4+ pro-B-ALL. Here, we explored the developmental impact of FLT3 activation alone, or together with MLL-AF4, in the hematopoietic fate of hESCs. FLT3 activation does not affect specification of hemogenic precursors but significantly enhances the formation of CD45(+) blood cells, and CD45(+)CD34(+) blood progenitors with clonogenic potential. However, overexpression of FLT3 mutations or wild-type FLT3 (FLT3-WT) completely abrogates hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly affects hESC specification rather than proliferation or survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling of hESC-derived CD45(+) cells supports the FLT3-mediated inhibition of hematopoiesis in MLL-AF4-expressing hESCs, which is associated with large transcriptional changes and downregulation of genes involved in hematopoietic system development and function. Importantly, FLT3 activation does not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells, suggesting the need of alternative (epi)-genetic cooperating hits This work was funded by FIS/FEDER (PI10/00449 to P.M. and PI11/00119 to C.B.) and by The Spanish Association Against Cancer to P.M. D.R.-M. is supported by PFIS scholarship (FI11/ 00511). C.B., P.J.R., and V.R.-M. are supported by “Miguel Servet” Fellowships (CP07/0059, CP09/0063, and CP12/03175). R.M. is supported by the ISCIII (CA10/01332). P.M. is an ICREA investigator supported by the ISCIII Red de Terapia Celular (Tercel, RD12/0019/0006). https://hdl.handle.net/10481/111105 Effects of polystyrene microbeads in marine planktonic crustaceans Gambardella, Chiara; Morgana, Silvia; Ferrando, Sara; Bramini, Mattia; Piazza, Veronica; Costa, Elisa; Garaventa, Francesca; Faimali, Marco Plastic debris accumulates in the marine environment, fragmenting into microplastics (MP), causing concern about their potential toxic effects when ingested by marine organisms. The aim of this study was to verify whether 0.1 µm polystyrene beads are likely to trigger lethal and sub-lethal responses in marine planktonic crustaceans. MP build-up, mortality, swimming speed alteration and enzyme activity (cholinesterases, catalase) were investigated in the larval stages of Amphibalanus amphitrite barnacle and of Artemia franciscana brine shrimp exposed to a wide range of MP concentrations (from 0.001 to 10 mg L-1) for 24 and 48 h. The results show that MP were accumulated in crustaceans, without affecting mortality. Swimming activity was significantly altered in crustaceans exposed to high MP concentrations (> 1 mg L-1) after 48 h. Enzyme activities were significantly affected in all organisms exposed to all the above MP concentrations, indicating that neurotoxic effects and oxidative stress were induced after MP treatment. These findings provide new insight into sub-lethal MP effects on marine crustaceans. This work has been supported by RITMARE (Ricerca Italiana per il MARE) Flagship Project, a National Research Program funded by the Italian Ministry of University and Research (MIUR) and by EPHEMARE (Ecotoxicological effects of microplastics in marine ecosystems) Project, a JPI Oceans.