Biology subjects

Stirparo, G. G.

Publications and source records attributed to Stirparo, G. G..

11 recordsLinked to original sources

bioRxiv2025

NETSseq Reveals Inflammatory and Aging Mechanisms in Distinct Cell Types Driving Cerebellar Decline in Ataxia Telangiectasia

The cellular and molecular changes driving the neurological abnormalities associated with ataxia - telangiectasia (A-T) are not well understood. Here, we applied our proprietary Nuclear Enriched Transcript Sort sequencing (NETSseq) platform to investigate changes in cell type composition and gene expression in human cerebellar post-mortem tissue from A-T and control donors. Compared to single-nuclei technologies, NETSseq provided a more robust detection of genes with low abundance, a higher cell type specific expression pattern, and significantly lower levels of cross-contamination. We found dysregulation in neurotransmitter signaling in granule neurons, potentially underlying the impaired motor coordination in A-T. Astrocytes and microglia have evidence of accelerated aging, with astrocytes being characterized by neurotoxic signatures, while microglia showed activation of DNA damage response pathways. These findings highlight the importance of NETSseq as a resource for investigating mechanisms and biological processes associated with disease, providing high-sensitivity, cell-specific insights to advance targeted therapies for neurodegenerative diseases.

neuroscience↗

bioRxiv2020

Capture of mouse and human stem cells with features of formative pluripotency

Pluripotent cells emerge via a naive founder population in the blastocyst, acquire capacity for germline and soma formation, and then undergo lineage priming. Mouse embryonic stem (ES) cells and epiblast stem cells (EpiSCs) represent the initial naive and final primed phases of pluripotency, respectively. Here we investigate the intermediate formative stage. Using minimal exposure to specification cues, we expand stem cells from formative mouse epiblast. Unlike ES cells or EpiSCs, formative stem (FS) cells respond directly to germ cell induction. They colonise chimaeras including the germline. Transcriptome analyses show retained pre-gastrulation epiblast identity. Gain of signal responsiveness and chromatin accessibility relative to ES cells reflect lineage capacitation. FS cells show distinct transcription factor dependencies from EpiSCs, relying critically on Otx2. Finally, FS cell culture conditions applied to human naive cells or embryos support expansion of similar stem cells, consistent with a conserved attractor state on the trajectory of mammalian pluripotency.

developmental biology↗

bioRxiv2020

Cell surface fluctuations regulate early embryonic lineage sorting

In development, lineage segregation of multiple lineages must be coordinated in time and space. An important example is the mammalian inner cell mass (ICM), in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the foetus). The physical mechanisms that determine this spatial segregation between EPI and PrE are still poorly understood. Here, we identify an asymmetry in cell-cell affinity, a mechanical property thought to play a significant role in tissue sorting in other systems, between EPI and PrE precursors (pEPI and pPrE). However, a computational model of cell sorting indicated that these differences alone appeared insufficient to explain the spatial segregation. We also observed significantly greater surface fluctuations in pPrE compared to pEPI. Including the enhanced surface fluctuation in pPrE in our simulation led to robust cell sorting. We identify phospho-ERM regulated membrane tension as an important mediator of the increased surface fluctuations in pPrE. Using aggregates of engineered cell lines with different surface fluctuation levels cells with higher surface fluctuations were consistently excluded to the outside of the aggregate. These cells behaved similarly when incorporated in the embryo. Surface fluctuations-driven segregation is reminiscent of activity-induced phase separation, a sorting phenomenon in colloidal physics. Together, our experiments and model identify dynamic cell surface fluctuations, in addition to static mechanical properties, as a key factor for orchestrating the correct spatial positioning of the founder embryonic lineages.

developmental biology↗

bioRxiv2020

Cooperative molecular networks drive a mammalian cell state transition

In the mammalian embryo, epiblast cells must exit their naive state and acquire formative pluripotency. This cell state transition is recapitulated by mouse embryonic stem cells (ESCs), which undergo pluripotency progression in defined conditions in vitro. However, our understanding of the molecular cascades and gene-networks involved in the exit from naive pluripotency remains fragmented. Here we employed a combination of genetic screens in haploid ESCs, CRISPR/Cas9 gene disruption, large-scale transcriptomics and computational systems-biology to delineate the regulatory circuits governing naive state exit. Transcriptome profiles for 73 knockout ESC lines predominantly manifest delays on the trajectory from naive to formative epiblast. We find that gene networks operative in ESCs are active during transition from pre- to post-implantation epiblast in utero. We identified 374 naive-associated genes tightly connected to epiblast state and largely conserved in human ESCs and primate embryos. Integrated analysis of mutant transcriptomes revealed funneling of multiple gene activities into discrete regulatory modules. Finally, we delineate how intersections with signaling pathways direct this pivotal mammalian cell state transition.

systems biology↗

bioRxiv2020

Trophectoderm Potency is Retained Exclusively in Human Naïve Cells

Classical mouse embryology has established a paradigm of early development driven by sequential lineage bifurcations. Accordingly, mouse embryonic stem cells derived from early epiblast have lost the potency to produce extraembryonic trophectoderm. We show in contrast that human naive epiblast cells readily make trophectoderm. Inhibition of ERK signalling, instrumental in naive stem cell propagation, unexpectedly potentiates trophectoderm formation, an effect enhanced by Nodal inhibition. Transcriptome analyses authenticate conversion into trophectoderm with subsequent production of syncitiotrophoblast, cytotrophoblast and trophoblast stem cells. Genetic perturbations indicate that NANOG suppresses and TFAP2C enables trophectoderm induction. Consistent with post-implantation progression, trophectoderm potential is extinguished in conventional human pluripotent stem cells, which instead make amnion. Finally, human embryo epiblasts from late blastocysts efficiently generate trophectoderm and differentiated trophoblast. Thus, pluripotent cells in the human embryo retain extraembryonic lineage plasticity and regenerative potential until implantation. Harnessing this unanticipated regulative capacity may be beneficial for assisted reproduction technology.

developmental biology↗

bioRxiv2020

Oct4 regulates embryonic pluripotency via metabolic mechanisms and Stat3 signalling.

OCT4 is a fundamental component of the molecular circuitry governing pluripotency in vivo and in vitro. To determine how OCT4 protects the pluripotent lineage from differentiation into trophoblast, we used single cell transcriptomics and quantitative immunofluorescence on blastocysts and established differentially expressed genes and pathways between control and OCT4 null cells. Activation of most pluripotency-associated transcription factors in the early mouse inner cell mass appears independent of OCT4, whereas JAK/STAT signalling requires OCT4, via activation of IL6ST. Single cell deconvolution, diffusion component and trajectory inference dissected the process of differentiation of OCT4 null cells by activating specific gene-network and transcription factors. Downregulation of glycolytic and oxidative metabolism was observed. CHIPseq analysis suggests OCT4 directly targets rate-limiting glycolytic enzymes. Concomitant with significant disruption of the STAT3 pathway, oxidative respiration is significantly diminished in OCT4 null cells. Upregulation of the lysosomal pathway detected in OCT4 null embryos is likely attributable to aberrant metabolism. Highlights and noveltyO_LIMajor pluripotency-associated transcription factors are activated in OCT4-deficient early mouse ICM cells, coincident with ectopic expression of trophectoderm markers C_LIO_LIJAK/STAT signalling is defective in OCT4 null embryos C_LIO_LIOCT4 promotes expression of KATS enzymes by means of glycolytic production of Acetyl CoA to secure chromatin accessibility for acquisition of epiblast identity C_LIO_LIOCT4 regulates the metabolic and biophysical processes required for establishment of embryonic pluripotency C_LI

developmental biology↗

bioRxiv2020

Sox2 modulation increases naive pluripotency plasticity

Induced pluripotency provides a tool to explore the molecular mechanisms underlying the establishment, maintenance and differentiation of naive pluripotent stem cells (nPSCs). Here, we report that self-renewal of nPSCs requires minimal Sox2 expression (Sox2-low). Sox2-low nPSCs do not show impaired neuroectoderm specification and differentiate efficiently in vitro into all embryonic germ lineages. Strikingly, Sox2-low cells also differentiate towards the trophoblast lineage both in vitro and in vivo. At the single-cell level self-renewing Sox2-low nPSCs exhibit a homogeneous naive molecular signature. However, they also display a basal trophoblast molecular signature and decreased ability of Oct4 to bind naive-associated regulatory sequences compared to control cells. These features underlie observed enhanced cell potency upon the removal of self-renewing cues. In sum, this work defines Sox2 as a restrictor of developmental potential and suggests perturbation of the naive pluripotent network as an underlying cause of increased cell potency. HighlightsO_LILow Sox2 expression is sufficient for naive pluripotent stem cell self-renewal C_LIO_LILow Sox2 expression does not impair neurectoderm differentiation in vitro C_LIO_LILow Sox2 expression impairs Oct4 genomic occupancy C_LIO_LILow Sox2 expression increases naive pluripotent cell plasticity in vitro and in vivo C_LI

developmental biology↗

bioRxiv2019

StemBond hydrogels optimise the mechanical microenvironment for embryonic stem cells

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and tethering of extracellular matrix (ECM) to substrates, making ECM tethering a potentially confounding variable in mechanical signalling investigations. Moreover, poor ECM tethering can lead to weak cell attachment. To address this, we developed StemBond hydrogels, a hydrogel formulation in which ECM tethering is stable and can be varied independently of stiffness. We show that soft StemBond hydrogels provide an optimal format for culturing embryonic stem (ES) cells. We find that soft StemBond substrates improve the homogeneity of ES cell populations, boost their self-renewal, and increase the efficiency of cellular reprogramming. Our findings underline how soft microenvironments impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

cell biology↗

bioRxiv2019

Efficient RNA-mediated reprogramming of human somatic cells to naive pluripotency facilitated by tankyrase inhibition

In contrast to conventional human pluripotent stem cells (hPSC) that are related to post-implantation embryo stages, naive hPSC exhibit features of pre-implantation epiblast. Naive hPSC are established by resetting conventional hPSC, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that tankyrase inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSC can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene free naive hPSC offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSC.

developmental biology↗

bioRxiv2018

Single-cell transcriptome analysis of human, marmoset and mouse embryos reveals common and divergent features of preimplantation development

The mouse embryo is the canonical model for mammalian preimplantation development. Recent advances in single-cell profiling allow detailed analysis of embryogenesis in other eutherian species, including human, to distinguish conserved from divergent regulatory programs and signalling pathways in the rodent paradigm. Here, we identify and compare transcriptional features of human, marmoset and mouse embryos by single-cell RNA-seq. Zygotic genome activation correlates with the presence of Polycomb Repressive Complexes in all three species, while ribosome biogenesis emerges as a predominant attribute in primate embryos, supporting prolonged translation of maternally deposited RNAs. We find that transposable element expression signatures are species-, stage- and lineage-specific. The pluripotency network in the primate epiblast lacks certain regulators operative in mouse, but encompasses WNT components and genes associated with trophoblast specification. Sequential activation of GATA6, SOX17 and GATA4 markers of primitive endoderm identity is conserved in primates. Unexpectedly, OTX2 is also associated with primitive endoderm specification in human and nonhuman primate blastocysts. Our cross-species analysis demarcates both conserved and primate-specific features of preimplantation development and underscores the molecular adaptability of early mammalian embryogenesis.

developmental biology↗

bioRxiv2017

Integrated analysis of single-cell embryo data yields a unified transcriptome signature for the human preimplantation epiblast

Single-cell profiling techniques create opportunities to delineate cell fate progression in mammalian development. Recent studies provide transcriptome data from human preimplantation embryos, in total comprising nearly 2000 individual cells. Interpretation of these data is confounded by biological factors such as variable embryo staging and cell-type ambiguity, as well as technical challenges in the collective analysis of datasets produced with different sample preparation and sequencing protocols. Here we address these issues to assemble a complete gene expression time course spanning human preimplantation embryogenesis. We identify key transcriptional features over developmental time and elucidate lineage-specific regulatory networks. We resolve post hoc cell-type assignment in the blastocyst, and define robust transcriptional prototypes that capture epiblast and primitive endoderm lineages. Examination of human pluripotent stem cell transcriptomes in this framework identifies culture conditions that sustain a naive state pertaining to the inner cell mass. Our approach thus clarifies understanding both of lineage segregation in the early human embryo and of in vitro stem cell identity, and provides an analytical resource for comparative molecular embryology.

developmental biology↗