João Pedro Agostinho de Sousa

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Agostinho de Sousa

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João Pedro

ORCID

0000-0001-8124-6624

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Publications 1 - 9 of 9

Epigenetic dynamics during capacitation of naïve human pluripotent stem cells
Agostinho de Sousa, João Pedro; Wong, Chee Wai; Dunkel, Ilona; et al. (2023)
Science Advances
Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.
Inhibition of PRC2 enables self-renewal of blastoid-competent naive pluripotent stem cells from chimpanzee
Huang, Tao; Radley, Arthur; Yanagida, Ayaka; et al. (2025)
Cell Stem Cell
Naive pluripotent stem cells (PSCs) are counterparts of early epiblast in the mammalian embryo. Mouse and human naive PSCs differ in self-renewal requirements and extraembryonic lineage potency. Here, we investigated the generation of chimpanzee naive PSCs. Colonies generated by resetting or reprogramming failed to propagate. We discovered that self-renewal is enabled by inhibition of Polycomb repressive complex 2 (PRC2). Expanded cells show global transcriptome proximity to human naive PSCs and embryo pre-implantation epiblast, with shared expression of a subset of pluripotency transcription factors. Chimpanzee naive PSCs can transition to multilineage competence or can differentiate into trophectoderm and hypoblast, forming tri-lineage blastoids. They thus provide a higher primate comparative model for studying pluripotency and early embryogenesis. Genetic deletions confirm that PRC2 mediates growth arrest. Further, inhibition of PRC2 overcomes a roadblock to feeder-free propagation of human naive PSCs. Therefore, excess deposition of chromatin modification H3K27me3 is an unexpected barrier to naive PSC self-renewal.
H3K18 lactylation marks tissue-specific active enhancers
Galle, Eva; Wong, Chee-Wai; Ghosh, Adhideb; et al. (2022)
Genome Biology
Background: Histone lactylation has been recently described as a novel histone post-translational modification linking cellular metabolism to epigenetic regulation. Results: Given the expected relevance of this modification and current limited knowledge of its function, we generate genome-wide datasets of H3K18la distribution in various in vitro and in vivo samples, including mouse embryonic stem cells, macrophages, adipocytes, and mouse and human skeletal muscle. We compare them to profiles of well-established histone modifications and gene expression patterns. Supervised and unsupervised bioinformatics analysis shows that global H3K18la distribution resembles H3K27ac, although we also find notable differences. H3K18la marks active CpG island-containing promoters of highly expressed genes across most tissues assessed, including many housekeeping genes, and positively correlates with H3K27ac and H3K4me3 as well as with gene expression. In addition, H3K18la is enriched at active enhancers that lie in proximity to genes that are functionally important for the respective tissue. Conclusions: Overall, our data suggests that H3K18la is not only a marker for active promoters, but also a mark of tissue specific active enhancers.
Characterization and modelling of the epigenetic dynamics during the transition from naïve to primed pluripotency
Agostinho de Sousa, João Pedro (2023)
Human pluripotent stem cells (hPSCs) show great potential for regenerative medicine applications, but several limitations must be addressed before their widespread clinical use. These limitations include epigenetic instability, which can lead to spontaneous differentiation or genomic instability, and heterogeneity among hPSC lines, resulting in cells with different and unexpected properties. In addition, there are safety concerns regarding the potential for teratoma formation, immune rejection, and prolonged culture of hPSCs–which may result in X chromosome-linked erosion, referring to the gradual loss of the silencing of one of the two X chromosomes in female cells. This erosion can cause increased expression of X-linked genes, affecting developmental and physiological consequences that can limit their differentiation potential and, consequently, their therapeutic efficacy. To overcome these limitations, it is necessary to improve our understanding of the genetic and epigenetic mechanisms that regulate hPSCs and develop culture conditions that more closely resemble the in vivo environment. Recent advances in human naïve culture conditions offer potential solutions for these challenges. Naïve human pluripotent stem cells maintained in culture resemble pre-implantation epiblast cells and represent an earlier developmental state than conventionally cultured hPSCs, which are in a primed state of pluripotency and resemble post-implantation epiblast cells. Compared to naïve cells, conventional hPSCs have a more restricted developmental potential and are closer to differentiation. We recently developed new culture conditions that allow naïve hPSCs to transition to the primed state of pluripotency within a time frame that mimics early development. We refer to this transition as "capacitation". However, it is uncertain whether capacitated naïve hPSCs can accurately replicate the gene expression and epigenetic patterns observed in the primed state after acquiring multi-lineage differentiation ability. Furthermore, it is unknown whether resetting conventionally cultured primed hPSCs to the naïve state and then allowing them to capacitate in the primed state could potentially address epigenetic anomalies such as X-chromosome inactivation erosion. Aiming to clarify these questions, we employed sequencing assays to characterize and model gene expression, DNA methylation, histone modifications, and chromatin accessibility landscapes of naïve, capacitated, and long-term cultured hPSCs. This dissertation presents and interprets the results of this multi-omics analysis, intended to enhance our understanding of the role of epigenetics in pluripotency, identify new targets for future research, and determine the advantages of naïve hPSCs capacitation. The results show that resetting conventionally cultured primed hPSCs to the naïve state followed by repriming restores the epigenetic characteristics of X chromosome inactivation from an eroded landscape. Additionally, they emphasise the importance of CpG islands, enhancers, and retrotransposons as hotspots of epigenetic dynamics between pluripotency states. Finally, they highlight the putative role of H3K27ac modifications in promoters of genes with naïve-specific expression. This study underscores the potential of improved hPSC culture methods for developing novel regenerative therapies and provides insights into the molecular mechanisms underlying epigenetic changes during capacitation. These findings have implications for generating clinically relevant cell types and contribute to the ongoing progress in regenerative medicine, laying the foundation for future investigations in optimizing human pluripotent stem cell culture methods.
Comparative analysis of mouse strains for in vivo induction of reprogramming factors
Picó, Sara; Vílchez-Acosta, Alba; Agostinho de Sousa, João Pedro; et al. (2025)
Cell Reports
In vivo reprogramming through the forced expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) has demonstrated great potential for reversing age-associated phenotypes. However, continuous in vivo OSKM expression has raised safety concerns due to loss of cell identity, decrease in body weight, and premature death. Although cyclic short-term or targeted expression of the reprogramming factors can mitigate some of these detrimental effects, systemic rejuvenation of wild-type mice has remained elusive. To improve the fundamental understanding of in vivo reprogramming, we conduct a comparative analysis of various reprogrammable mouse strains across multiple tissues and organs. In addition, we develop reprogrammable mouse strains by avoiding OSKM expression in specific organs or implementing expression approaches within specific cells, thereby offering safer strategies to induce in vivo reprogramming. We hope that these tools will become valuable resources for future research in this field of research with potential implications to human health.
scEpiAge: an age predictor highlighting single-cell ageing heterogeneity in mouse blood
Bonder, Marc Jan; Clark, Stephen J.; Krueger, Felix; et al. (2024)
Nature Communications
Ageing is the accumulation of changes and decline of function of organisms over time. The concept and biomarkers of biological age have been established, notably DNA methylation-based clocks. The emergence of single-cell DNA methylation profiling methods opens the possibility of studying the biological age of individual cells. Here, we generate a large single-cell DNA methylation and transcriptome dataset from mouse peripheral blood samples, spanning a broad range of ages. The number of genes expressed increases with age, but gene-specific changes are small. We next develop scEpiAge, a single-cell DNA methylation age predictor, which can accurately predict age in (very sparse) publicly available datasets, and also in single cells. DNA methylation age distribution is wider than technically expected, indicating epigenetic age heterogeneity and functional differences. Our work provides a foundation for single-cell and sparse data epigenetic age predictors, validates their functionality and highlights epigenetic heterogeneity during ageing.
Evaluating the connection between diet quality, EpiNutrient intake and epigenetic age: an observational study
Bordoni, Laura; Agostinho de Sousa, João Pedro; Zhuo, Jingran; et al. (2024)
The American Journal of Clinical Nutrition
Background: DNA methylation (DNAm) has unique properties which makes it a potential biomarker for lifestyle-related exposures. Epigenetic clocks, particularly DNAm-based biological age predictors [epigenetic age (EA)], represent an exciting new area of clinical research and deviations of EA from chronological age [epigenetic age acceleration (EAA)] have been linked to overall health, age-related diseases, and environmental exposures. Objectives: This observational study investigates the relationships between biological aging and various dietary factors within the LifeLines-DEEP Cohort. These factors include diet quality, processed food consumption, dietary glycemic load, and intake of vitamins involved in maintaining the epigenetic homeostasis (vitamins B-9, B-12, B-6, B-2, and C). Methods: Dietary records collected using food-frequency questionnaires were used to estimate diet quality [LifeLines Diet Score (LLDS)], measure the intake of unprocessed/ultraprocessed food according to the NOVA food classification system, and the adequacy of the dietary intake of vitamins B-9, B-12, B-2, B-6, and C. EA using Horvath, Hannum, Levine, and Horvath2 epigenetic clock models and DNAm-predicted telomere length (DNAm-TL) were calculated from DNAm data in 760 subjects. Associations between dietary factors and EAA were tested, adjusting for sex, energy intake, and body composition. Results: LLDS was associated with EAA (EAA_Horvath: β: −0.148; P = 1 × 10⁻⁴; EAA_Hannum: β: −0.148; P = 9 × 10⁻⁵; EAA_Levine: β: −0.174; P = 1 × 10⁻⁵; and EAA_Horvath2: β: −0.176; P = 4 × 10⁻⁶) and DNAm-TL (β: 0.116; P = 0.003). Particularly, EAA was associated with dietary glycemic load (EAA_Horvath: β: 0.476; P = 9 × 10⁻¹⁰; EAA_Hannum: β: 0.565; P = 1 × 10⁻¹³; EAA_Levine: β: 0.469; P = 5 × 10⁻⁹; EAA_Horvath2: β: 0.569; P = 1 × 10⁻¹³; and DNAmTL adjusted for age: β: −0.340; P = 2 × 10⁻⁵) and different measures of food processing (NOVA classes 1 and 4). Positive EAA was also associated with inadequate intake of vitamin B-12 (EAA_Horvath: β: −0.167; P = 0.002; EAA_Hannum: β: −0.144; P = 0.007; and EAA_Horvath2: β: −0.126; P = 0.019) and C (EAA_Hannum: β: −0.136; P = 0.010 and EAA_Horvath2: β: −0.151; P = 0.005). Conclusions: Our findings corroborate the hypothesis that nutrition plays a pivotal role in influencing epigenetic homeostasis, especially DNAm, thereby contributing to individual health trajectories and the pace of aging.
Developmental progression continues during embryonic diapause in the roe deer
Rüegg, Anna B.; van der Weijden, Vera A.; Agostinho de Sousa, João Pedro; et al. (2024)
Communications Biology
Embryonic diapause in mammals is a temporary developmental delay occurring at the blastocyst stage. In contrast to other diapausing species displaying a full arrest, the blastocyst of the European roe deer (Capreolus capreolus) proliferates continuously and displays considerable morphological changes in the inner cell mass. We hypothesised that developmental progression also continues during this period. Here we evaluate the mRNA abundance of developmental marker genes in embryos during diapause and elongation. Our results show that morphological rearrangements of the epiblast during diapause correlate with gene expression patterns and changes in cell polarity. Immunohistochemical staining further supports these findings. Primitive endoderm formation occurs during diapause in embryos composed of around 3,000 cells. Gastrulation coincides with elongation and thus takes place after embryo reactivation. The slow developmental progression makes the roe deer an interesting model for unravelling the link between proliferation and differentiation and requirements for embryo survival.
Metallomic profiles of pregnant women living with obesity in the UK: a secondary analysis of UPBEAT
Agostinho de Sousa, João Pedro; Griffiths, Alexander; Dalrymple, Kathryn V.; et al. (2025)
Metallomics
Characterization of serum metal element concentrations in pregnancy enables the elucidation of relationships with maternal-fetal and neonatal health. Metal elements in the blood serve as essential cofactors for enzymatic reactions and contribute to blood gas homeostasis, hormone synthesis, and physiological immune function for mother and fetus. Sub-optimal concentrations of some metals have been linked to adverse outcomes, including preterm birth, low birth weight, and impaired neurodevelopment. Maternal obesity also adversely influences metabolic status, including metal metabolism, with the potential for a heightened risk of complications at delivery and long-term health issues in offspring. Research on metal element levels in pregnant women with obesity and their effects on pregnancy outcomes is however limited. This study aims to characterize mid-gestation serum concentrations of 18 metal elements in samples from 755 pregnant women with obesity enrolled in the UK Pregnancies Better Eating and Activity Trial (UPBEAT) and identify associations with pregnancy outcomes. We found that calcium concentration tended to decrease with increasing parity, with an estimated reduction of 6.03 mg/L in multiparous participants compared to nulliparous participants (95% CI: -9.50 to -2.57 mg/L, P = 0.001). Additionally, elevated manganese concentrations at mid-pregnancy were associated with an increased incidence of antepartum haemorrhage after 34 weeks (OR: 4.62, 95% CI: 2.06-12.4, P < 0.001), and higher maternal phosphorus levels were linked to neonatal intensive care unit admissions (OR: 2.83, 95% CI: 1.75-4.67, P < 0.001). A future focus on dysregulation of these metal elements is needed to improve understanding of the clinical associations observed.

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João Pedro Agostinho de Sousa

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