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Chuva de Sousa Lopes, S. M.

Publications and source records attributed to Chuva de Sousa Lopes, S. M..

10 recordsLinked to original sources

The immediate cellular response to whole-genome doubling is conserved across polyploid contexts

Polyploid cells, which contain more than two copies of the genome, are widely present across plants and animals, where they are often found in tissues with high biosynthetic and metabolic demands, such as the mammalian liver and placenta. While somatic polyploidy is frequently associated with increased cell growth and biosynthetic capacity, unscheduled polyploidization in cell types that are not normally programmed to become polyploid is often linked to reduced cellular fitness and genome instability. To understand whether these divergent outcomes stem from distinct immediate cellular responses to increased ploidy, we systematically compared the early consequences of polyploidization across naturally occurring and experimentally induced systems. Specifically, we examined physiological polyploid cells in the Caenorhabditis elegans intestine and human hepatocyte organoids, alongside unscheduled polyploid human retinal pigment epithelial (RPE1) cells generated through cytokinesis failure. Using quantitative imaging, flow cytometry, and FUCCI-based cell-cycle reporters we measured cell size and protein translation dynamics during G1 in diploid and polyploid cells. Across all systems, we observed a strikingly conserved relationship between ploidy, cell size, and biosynthetic capacity: both cell size and protein translation showed similar scaling patterns after polyploidization, regardless of whether polyploidization occurred as part of normal development or by inducing cytokinesis failure. These findings indicate that the immediate cellular response to increased ploidy is broadly similar across contexts. However, in contrast to unscheduled polyploid RPE1 cells, polyploid human hepatocytes extend their G1 phase, leading to a higher accumulation of proteins before cell-cycle progression. Together, our findings suggest that polyploidization elicits similar growth responses across contexts, and that cell-type specific cell-cycle adaptations may determine whether polyploidy becomes advantageous or deleterious.

cell biology↗

The pro-inflammatory cytokines IFN-α and TNF-α inhibit organoid-derived extravillous trophoblast invasion

Proper placental development requires differentiation and invasion of extravillous trophoblasts (EVTs) into the maternal decidua to ensure adequate remodeling of spiral arteries and support fetal growth. Perturbations in these processes are associated with pregnancy complications such as preeclampsia and fetal growth restriction. The incidence of these pregnancy complications is increased in women with immune-mediated inflammatory diseases, which are characterized by elevated levels of pro-inflammatory cytokines, such as interferon alpha (IFN-) and tumor necrosis factor alpha (TNF-). However, the direct effects of these cytokines on trophoblast differentiation and invasion remain unclear. Using human trophoblast organoid models, we demonstrate that IFN- and TNF- impair EVT invasion while preserving their differentiation capacity. High-resolution imaging of untreated organoid-decidua co-cultures revealed extensive invasion of organoid-derived trophoblasts into the decidual stroma and spiral arteries. In contrast, organoids treated with IFN-, in particular, exhibited markedly reduced invasion within this system. Transcriptome profiling indicated altered expression of several pathways involved in invasion upon cytokine treatment. These findings support the notion that elevated levels of IFN- and TNF- can directly impair trophoblast invasive capacity, potentially contributing to suboptimal placental development and adverse pregnancy outcomes in the context of inflammatory disorders.

developmental biology↗

Reconstitution of human fetal ovaries reveals niche requirements for primordial germ cell-like cell progression

Human primordial germ cell-like cells (hPGCLCs) can be specified from human induced pluripotent stem cells (hiPSCs), offering a valuable model for human germ cell development. However, further maturation steps of hPGCLCs rely on mouse feeders, or co-culture with mouse gonadal somatic cells. Exposure of hPGCLCs to human embryonic niche has not been attempted. Here, we co-cultured female hPGCLCs in two distinct somatic compartments. In reconstituted ovary (rOv) culture, human fetal germ cells proliferate and initiate meiosis, while hPGCLCs upregulate gonadal germ cell markers such as DDX4. Additionally, hPGCLCs can be supported in 3D culture by the amnion-like cells (AMLC) generated during PGCLC differentiation. Compared to rOV, hPGCLCs are less prone to dedifferentiation in PGCLC/AMLC aggregates. Finally, we demonstrated that SCF is crucial for the survival of hPGCLCs but not second trimester fetal germ cells. Together, this work highlights a shift in niche is required in human germ cell development. In BriefChang and colleagues utilized in vitro reconstituted human fetal ovary (rOv) as somatic niche to mature human primordial germ cell-like cells (PGCLCs). hPGCLCs in rOv upregulate gonadal germ cell markers but are prone to dedifferentiation. In contrast, hPGCLCs cultured with amnion-like cells can be maintained without dedifferentiation. In both culture systems, SCF is crucial for the survival of hPGCLCs. HighlightsO_LIReconstituted human fetal ovaries (rOvs) support meiosis entry of fetal germ cells C_LIO_LIThe rOVs support hPGCLCs to upregulate gonadal germ cell markers C_LIO_LIhPGCLCs show less dedifferentiation in amnion-like cell aggregates compare to rOv C_LIO_LISCF is not required for survival of fetal germ cells, but crucial for hPGCLCs C_LI

developmental biology↗

An engineered tumor organoid model reveals cellular identity and signaling trajectories underlying translocation RCC.

Translocation renal cell carcinoma (tRCC) is a rare, aggressive type of kidney cancer primarily occurring in children. They are genetically defined by translocations involving MiT/TFE gene family members, TFE3 or, in rare cases, TFEB. The biology underlying tRCC development remains poorly understood, partly due to the lack of representative experimental models. Here, we utilized human kidney organoids, or tubuloids, to engineer a tRCC model by expression of one of the most common MiT/TFE fusions, SFPQ-TFE3. Fusion expressing tubuloids adopt a tRCC-like phenotype and gene expression signature in vitro and grow as clear cell RCC upon xenotransplantation in mice. Genome-wide binding analysis reveals that SFPQ-TFE3 reprograms gene expression signatures by aberrant, gain-of-function genome-wide DNA binding. Combining these analyses with single-cell mRNA readouts reveals an epithelium-to-mesenchymal differentiation trajectory underlying tRCC transformation, potentially caused by deregulated Wnt signaling. Our study demonstrates that SFPQ-TFE3 expression is sufficient to transform kidney epithelial cells into tRCC and defines the trajectories underlying malignant transformation, thereby facilitating the development of new therapeutic interventions.

cancer biology↗

Binucleated human hepatocytes arise through loss of membrane anchorage to the midbody during endomitosis

Many plant and animal cells transition from canonical to non-canonical cell cycles during development, resulting in the formation of polyploid cells. Two types of non-canonical cell cycles exist: endoreplication, where cells increase their DNA content without entering M phase, and endomitosis, where cells enter M phase but exit prematurely. Although endoreplication has been extensively studied in plants and insects, much less is known on the regulation of endomitosis, which is the most common mode of polyploidization in mammals. In this study, we use fetal-derived human hepatocyte organoids (Hep-Org), to investigate how human hepatocytes initiate and execute endomitosis. We find that cells in endomitosis M phase have normal mitotic timings, but lose membrane anchorage to the midbody during cytokinesis, resulting in regression of the cytokinetic furrow and formation of binucleate cells. Using immunofluorescence, we find that three cortical anchoring proteins, RacGAP1, anillin, and citron kinase (CIT-K), lose their association with the cell cortex during cytokinetic regression. Moreover, reduction of WNT activity by withdrawal of CHIR99021, a GSK3 inhibitor, from the culturing medium increases the percentage of binucleated cells in Hep-Orgs. This effect is lost in organoids with mutations in the atypical E2F proteins, E2F7 and E2F8, which have been implicated in binucleation of rodent hepatocytes. Together, our results identify how human hepatocytes inhibit cell division in endomitosis, and highlight an evolutionary recurrent mechanism to initiate non-canonical cell cycles in mammals.

cell biology↗

Efficient and scalable generation of primordial germ cells in 2D culture using basement membrane extract overlay

Current human primordial germ cell like cells (hPGCLCs) differentiation methods from human pluripotent stem cells (hPSCs) are inefficient, and it is challenging to generate sufficient hPGCLCs to optimize in vitro gametogenesis. We present a new differentiation method that uses diluted basement membrane extract (BME) and low BMP4 concentration to efficiently induce hPGCLC differentiation in scalable 2D cell culture. We show that BME overlay potentiated BMP/SMAD signaling, induced lumenogenesis and increased expression of key hPGCLC-progenitor markers such as TFAP2A and EOMES. These findings highlight the importance of (factors in) BME during hPGCLC differentiation, and demonstrate the potential of the BME-overlay method to interrogate the formation of PGCs and amnion in humans as well to investigate the next steps to achieve in vitro gametogenesis. HIGHLIGHTSO_LIhPGCLCs can be generated efficiently in 2D from hPSCs with treatment of BMP4 and BME overlay C_LIO_LIBME overlay method is highly scalable, cost-effective and simple to perform C_LIO_LIhPGCLCs differentiate together with amniotic ectoderm- and mesoderm-like cells from a TFAP2A+/CDX2+/EOMES+/GATA3+ common progenitor population C_LIO_LIBME overlay enables robust hPGCLC formation by potentiating BMP/SMAD signaling in the common progenitor population C_LI

developmental biology↗

Characterization of the human fetal rete region by single cell transcriptional analysis of gonads and mesonephros/epididymis

During development of the male reproductive tract, the human rete functions as a bridging structure between the seminiferous tubes and the efferent ducts of the epididymis. However, despite its significant contribution to human sex-specific gonadogenesis and future male fertility, the rete testis remains poorly understood. To investigate fetal rete testis development, we have performed single-cell transcriptomics on human fetal testes (and ovaries) and mesonephros/epididymis from first and second trimester. By revealing KRT19 and PAX8 as rete markers, we were able to identify the molecular signatures of the rete epithelial cells and mesonephros/epididymis epithelial cells. In the process, we have identified a population of Sertoli cells and germ cells in the rete testis. Moreover, we also revealed a small population of epithelial cells in the rete ovarii, comparable to the epithelial cells in the rete testis. Together, our study provides insights into human fetal sex-specific gonadogenesis and development of the male reproductive tract beyond the gonads.

developmental biology↗

A clusterability measure for single-cell transcriptomics reveals phenotypic subpopulations

The ability to discover new cell populations by unsupervised clustering of single-cell transcriptomics data has revolutionized biology. Currently, there is no principled way to decide, whether a cluster of cells contains meaningful subpopulations that should be further resolved. Here we present SIGMA, a clusterability measure derived from random matrix theory, that can be used to identify cell clusters with non-random sub-structure, testably leading to the discovery of previously overlooked phenotypes.

bioinformatics↗

Synthetic Maturation of Multilineage Human Liver Organoids via Genetically Guided Engineering

Pluripotent stem cell (PSC)-derived organoids are emerging as novel human-based microphysiological models but display immature phenotypes with limited subsets of endothelial or stromal cells. Here we demonstrate that in vitro manipulation of gene regulatory networks (GRNs) in PSC-derived liver organoids selected either through computational analysis or targeted tissue design can advance tissue maturation in vitro. Through an unbiased comparison with the genetic signature of mature livers, we identify downregulated GRNs in fetal liver organoids compared to adult livers. We demonstrate that overexpression of PROX1 and ATF5, together with targeted CRISPR-based transcriptional activation of endogenous CYP3A4, drives maturation in vitro. Single cell analyses reveal hepatobiliary-, endothelial-, and stellate-like cell populations. The engineered organoids demonstrate enhanced vasculogenesis, capture native liver characteristics (e.g. FXR signaling, CYP3A4 activity), and exhibit therapeutic potential in mice. Collectively, our approach provides a genetically guided framework for engineering developmentally advanced multilineage tissues from hiPSCs. HIGHLIGHTSO_LIIn vitro tissue maturation via genetically encoded molecular programs C_LIO_LIComputational analysis to identify maturation transcription factors in liver organoids C_LIO_LIPromoting vascularization of organoids via genetically encoded molecular programs C_LIO_LISingle cell analysis of parenchymal and non-parenchymal cells C_LIO_LIModeling of native liver functions and in vivo therapeutic potential C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=168 HEIGHT=200 SRC="FIGDIR/small/087445v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@6ec32aorg.highwire.dtl.DTLVardef@1cf2eeforg.highwire.dtl.DTLVardef@1128748org.highwire.dtl.DTLVardef@16fb5a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

DNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of terminally differentiated gametes to the pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.

genomics↗