Generation of pre-clinical cellular models for disease modeling
Creation of induced pluripotent stem cells (iPSCs) through Yamanaka reprogramming from patients with inherited genetic mutations causing neurological (e.g., Angelman syndrome) and cardiac diseases (e.g., hypertrophic cardiomyopathy - HCM) as well as predisposition to cancer (e.g., familiar breast cancer). Correction of mutations by genetic engineering approaches based on CRISPR/Cas9 technology to create isogenic iPSC control lines for disease modelling. Use of 2D and 3D models to study disease phenotypes through differentiation (e.g., neurons, cardiomyocytes, mammary epithelium) of mutated and corrected iPSCs. Modulation of expression of disease-related genes with sophisticated inducible CRISPR/Cas9-based systems (e.g., CRISPRa/CRISPRi) and degron systems.
Improving reprogramming protocols to generate clinical-grade iPSCs
Testing different reprogramming variables and culture conditions (e.g., donor cell source, medium conditions, oxygen concentrations) to generate iPSCs devoid of recurrent genetic and epigenetic errors. Epigenetic monitoring of genomic imprinting and X-chromosome inactivation as read-outs of epigenetic fidelity in iPSCs by molecular analysis. Quality control through multiple pluripotency tests and genomics/transcriptomics/epigenomics approaches.
Monitoring the effects of epigenetic dysfunction in iPSCs in their downstream applications
Testing the consequences of abnormal epigenetic states (e.g., X-chromosome erosion, imprinting dysfunction) in iPSCs on disease modelling and cell-replacement therapies. Transcriptional and functional analyses of iPSCs differentiated into neuronal, epithelial or cardiomyocyte lineages through confocal microscopy, single-cell transcriptomics and electrophysiological approaches. Implantation of iPSC-differentiated derivatives in model organisms and assessing the effect of epigenetic errors in disease recovery or tumorigenic potential.
Simão José Teixeira da Rocha, Stem Cell Epigenetics (Keywords: Stem cells, Epigenetics, Genomic Imprinting, X-inactivation, Disease modeling)
Evguenia Pavlovna Bekman Gaspar, 3D brain organoids for disease modelling and therapeutic purposes. (Keywords: Cerebellar organoids, neuronal differentiation of pluripotent stem cells, iPSCs, disease modeling, gene editing)
Miguel Casanova, Transposable elements in physiological and pathological contexts (Keywords: Human embryonic stem cells, Computational Biology, Transposable elements, non-coding RNA, CRISPR gene editing)
Samantha Mancino, Epigenetic signatures and brain aging (Keywords: Imprinting, Epigenetic Clock, Neurodegeneration, iPSC, Neuronal differentiation)
Ana Cláudia Bernardino Raposo, Consequences of the instability of X-chromosome inactivation in human induced Pluripotent Stem Cells (Keywords: XIST, X-inactivation, Dosage compensation, Erosion, female iPSCs)
Maria Arez, Epigenetic stability in iPSCs reprogramming (Keywords: Epigenetics, Genomic Imprinting, iPSCs, Reprogramming process, DNA methylation)
Carina Maranga, Generation of 3D cerebellar organoids to model Angelman Syndrome (Keywords: Cerebellar organoids, Angelman syndrome, Disease modeling)
Adriana Vieira, In vitro modeling of Angelman syndrome using the neural commitment of patient-specific iPSCs (Keywords: Angelman syndrome, genomic imprinting, iPSCs, neuronal differentiation, disease modeling)
Melanie Esckersley-Maslin (Univeristy of Melbourne), Wolf Reik (Babraham Institute), Jan Zylicz (Novo Nordisk Foundation Center for Stem Cell Biology), Edith Heard (EMBL), Nuno Barbosa-Morais (iMM), Maria Carmo-Fonseca (iMM), Bruno Bernardes de Jesus (iBIMED, Univ. Aveiro); Laura Steenplass (University Hospital Essen) Maud Borenzstein (IGMM, Montpellier)
Arez, M., Eckersley-Maslin, M., Klobučar, T., von Gilsa Lopes, J., Krueger, F., Raposo, A.C., Gendrel, A.-V., Bernardes de Jesus, B., da Rocha, S.T. Sex of donor cell and reprogramming conditions predict the extent and nature of imprinting defects in mouse iPSCs. bioRxiv doi: 10.1101/2020.11.20.370973 (2021).
Silva, T.P., Pereira, C.A., Raposo, A.C., Oliveira, A.R., Arez, M., Cabral, J.M.S., Milagre, I., Carmo-Fonseca, M., Rocha, S.T.D. Generation and characterization of induced pluripotent stem cells heterozygous for the Portuguese BRCA2 founder mutation. Stem Cell Res 53:102364 (2021).
Silva, T.P., Pereira, C.A., Oliveira, A.R., Raposo, A.C., Arez, M., Cabral, J.M.S., Milagre, I., Carmo-Fonseca, M., da Rocha, S.T. Generation and characterization of induced pluripotent stem cells from a family carrying the BRCA1 mutation c.3612delA. Stem Cell Res 52:102242 (2021).
Klobučar, T., Kreibich, E., Krueger, F., Arez, M., Pólvora-Brandão, D., von Meyenn, F., da Rocha, S.T., Eckersley-Maslin, M. IMPLICON: an ultra-deep sequencing method to uncover DNA methylation at imprinted genes. Nucleic Acids Res 48(16):e92 (2020).
Maranga, C., Fernandes, T.G., Bekman, E., da Rocha, S.T. Angelman syndrome: a journey through the brain. FEBS J 287(11):2154-2175 (2020).
da Rocha, S.T., Gendrel, A.V. The influence of DNA methylation on monoallelic expression. Essays Biochem 63(6):663-676 (2019).
Bousard, A., Raposo, A.C., Żylicz, J.J., Picard, C., Pires, V.B., Qi, Y., Gil, C., Syx, L., Chang, H.Y., Heard, E., da Rocha, S.T. The role of Xist-mediated Polycomb recruitment in the initiation of X-chromosome inactivation. EMBO Rep 20:e48019 (2019).
Pólvora-Brandão, D., Joaquim, M., Godinho, I., Aprile, D., Álvaro, A.R., Onofre, I., Raposo, A.C., Pereira de Almeida, L., Duarte, S.T., da Rocha, S.T. Loss of hierarchical imprinting regulation at the Prader-Willi/Angelman syndrome locus in iPSCs. Hum Mol Genet 27(23):3999-4011 (2018).