Biology subjects

Martello, G.

Publications and source records attributed to Martello, G..

16 recordsLinked to original sources

bioRxiv2026

Lifelong Invertebrate Chordate Central Nervous System Regeneration is Driven by a Migrating Pool of Conserved Neural Stem Cells Program

Adult central nervous systems (CNS) are generally constrained by limited cellular replacement. How regenerating organisms integrate structural maintenance with continuous, cyclic renewal remains unresolved. Here, we leverage the colonial chordate Botryllus schlosseri which completely resorbs and regenerates its entire CNS de novo weekly. Combining indexed single-cell sorting, Smart-seq3 sequencing, spatial mapping, and temporal deconvolution, our studies suggest that this continuous CNS renewal is mediated by an intrinsic pool of lineage-restricted neural stem cells. Single-cell analysis resolved a conserved SOX2/NOTCH1/NEUROG1 prospective progenitor continuum alongside mature excitability-defined neurons. Brain aging drives a phase-specific desynchronization of this regenerative program, inducing early hyper-activation followed by a sudden collapse. Finally, syngeneic transplantation and in vivo tracking show that neural-complex-derived cells persist after vascular delivery and localize to developing bud and neural-associated territories, with injury-associated accumulation near the neural gland. This work establishes a spatiotemporal framework for lifelong chordate CNS renewal and aging.

cell biology↗

bioRxiv2025

Feeder-free culture of naive human pluripotent stem cells retaining embryonic, extraembryonic and blastoid generation potential

Conventional human pluripotent stem cells (hPSCs) are widely used to study early embryonic development, generate somatic cells, and model diseases, with differentiation potential aligned to a post-implantation epiblast identity. In the past decade, naive hPSCs, representing a pre-implantation stage, have been derived. Naive hPSCs efficiently differentiate towards embryonic and extraembryonic lineages such as trophectoderm, primitive endoderm, and extraembryonic mesoderm, and also self-organize into blastocyst-like structures called blastoids. However, their culture typically relies on mouse embryonic fibroblasts (MEFs), which are variable, resource-intensive, and can confound analyses. We report the long-term maintenance of naive hPSCs in a feeder-free, serum-coated system. We successfully expanded for up to 25 passages 8 different naive hPSCs lines across 5 laboratories. Growth rate, clonogenicity, and gene expression profiles on serum coating were comparable to MEF-based cultures, but serum coating eliminated fibroblast contamination. Naive hPSCs cultured on serum exhibited more efficient germ layer specification, retained trophectoderm potential and high blastoid formation efficiency. Exome sequencing revealed fewer mutations in serum-cultured cells, and mass spectrometry identified extracellular matrix proteins such as vitronectin, fibronectin, and collagens in the serum coating. Overall, serum coating offers a scalable, cost-effective and therefore widely applicable alternative for naive hPSC culture, maintaining developmental potential, reducing DNA mutations, and eliminating MEF-related confounding factors. We believe serum coating will expand the use of naive hPSCs to large-scale studies and facilitate the investigation of mechanistic insights into developmental and disease modelling.

cell biology↗

bioRxiv2024

Unveiling Gene Perturbation Effects through Gene Regulatory Networks Inference from single-cell transcriptomic data

1Physiological and pathological processes are governed by a network of genes called gene regulatory networks (GRNs). By reconstructing GRNs, we can accurately model how cells behave in their natural state and predict how genetic changes will affect them. Transcriptomic data of single cells are now available for a wide range of cellular processes in multiple species. Thus, a method building predictive GRNs from single-cell RNA sequencing (scRNA-seq) data, without any additional prior knowledge, could have a great impact on our understanding of biological processes and the genes playing a key role in them. To this aim, we developed IGNITE (Inference of Gene Networks using Inverse kinetic Theory and Experiments), an unsupervised machine learning framework designed to infer directed, weighted, and signed GRNs directly from unperturbed single-cell RNA sequencing data. IGNITE uses the GRNs to generate gene expression data upon single and multiple genetic perturbations. IGNITE is based on the inverse problem for a kinetic Ising model, a model from statistical physics that has been successfully applied to biological networks. We tested IGNITE on murine pluripotent stem cells (PSCs) transitioning from the naive to formative states. Using as input only scRNA-seq data of unperturbed PSCs, IGNITE simulated single and triple gene knockouts. Comparison with experimental data revealed high accuracy, up to 74%, outperforming currently available methods. In sum, IGNITE identifies predictive GRNs from scRNA-seq data without additional prior knowledge and faithfully simulates single and multiple gene perturbations. Applications of IGNITE range from studying cell differentiation to identifying genes specifically active under pathological conditions.

systems biology↗

bioRxiv2023

TGF-beta dynamically controls epithelial identity in a 3D model of human epiblast

Pluripotency is the ability to give rise to all cell types of the body and is first observed in a mass of disorganised cells of the embryo. Upon implantation, pluripotent cells form a columnar epithelium and undergo lumenogenesis. At gastrulation, a portion of the pluripotent epiblast will undergo epithelial to mesenchymal transition (EMT), forming the primitive streak (PS). It still remains unclear what molecular mechanism supports the epithelial identity of the pluripotent epiblast before gastrulation. Here we developed an optimised, chemically defined 3D model of human pluripotent epiblast formation in which conventional pluripotent stem cells (PSCs) self-organise into a columnar epithelium with a lumen in 48 hours. From 72 hours we observed spontaneous symmetry breaking and specification of PS-like cells, as confirmed by single-cell RNA sequencing. We found that Insulin and FGF signalling are both required for the proliferation and survival of the pluripotent epiblast model. Conversely, TGF-beta signalling maintains epithelial identity. Epithelial identity appears uncoupled from the expression of canonical pluripotency markers OCT4, NANOG and PRDM14, but under the control of ZNF398. Once the pluripotent epithelium is established, TGF-beta inhibition is inconsequential, and stimulation with Activin A leads to highly efficient PS induction. We conclude that TGF-beta dynamically orchestrates epithelial identity of human pluripotent cells.

developmental biology↗

bioRxiv2023

KLF7 is a general inducer of human pluripotency

Pluripotency is the capacity to give rise to all differentiated cells of the body and the germ line and is governed by a self-reinforcing network of transcription factors. The forced expression of only some of these factors enables the reprogramming of somatic cells to pluripotency. In murine cells, several kruppel-like factors (KLFs) have been identified as stabilisers and inducers of pluripotency. Human somatic cells are routinely reprogrammed by expression of KLF4 in combination with OCT4, SOX2 and cMYC (OSKM). An extensive transcriptome analysis revealed, however, that KLF4 is barely expressed in conventional human pluripotent stem cells (PSCs). Here we show that KLF7 is robustly expressed in conventional human PSCs and it allows transcription factor-mediated somatic reprogramming. Moreover, we find that KLF7 is highly expressed in naive PSCs and its forced expression in conventional hPSCs induces upregulation of naive markers and boosts efficiency of chemical resetting to naive PSCs, overall suggesting that KLF7 is a general human pluripotency factor and an inducer of pluripotency.

developmental biology↗

bioRxiv2023

New permanent stem cell niche for development and regeneration in a chordate

Stem cell niches are defined as the microenvironments where stem cells home, receiving stimuli defining their fate. In vertebrates, stem cell niches are stable and physically confined compartments. Botryllus schlosseri is an invertebrate colonial chordate where temporary stem cell niches have been identified in adult individuals that are cyclically resorbed and replaced by a new generation of clonal zooids. B. schlosseri also displays remarkable regenerative abilities, being capable of whole-body regeneration, but the cellular source of these processes is still unknown. Here we identified by means of a high-resolution morphological characterization a new putative stem cell niche in the ampullae of the circulatory system acting as a stem cell source during asexual reproduction. Stem cells of the ampullae travel via the circulatory system and contribute to the development of several organs and could explain where stem cells contributing to whole-body regeneration are stored. The ampullae niches are stable during the life cycle and regeneration of B. schlosseri, while additional niches of the zooid are dynamically established and colonised by circulating stem cells. Our results reveal an unprecedented dynamicity of stem cell niches in highly regenerative invertebrates.

cell biology↗

bioRxiv2023

Identification of druggable host dependency factors shared by multiple SARS-CoV-2 variants of concern

The high mutation rate of SARS-CoV-2 leads to emergence of several variants, some of which are resistant to vaccines and drugs targeting viral elements. Targeting host dependency factors - cell proteins required for viral replication - would help avoid resistance. However, whether different SARS-CoV-2 variants induce conserved cell responses and exploit the same core host factors is still unclear. We compared three variants of concern and observed that the host transcriptional response was conserved, differing only in kinetics and magnitude. By CRISPR screening we identified the host genes required for infection by each variant: most of the identified genes were shared by multiple variants, both in lung and colon cells. We validated our hits with small molecules and repurposed FDA-approved drugs. All drugs were highly effective against all tested variants, including delta and omicron, new variants that emerged during the study. Mechanistically, we identified ROS production as a pivotal step in early virus propagation. Antioxidant drugs, such as N-acetyl cysteine (NAC), were effective against all variants both in human lung cells, and in a humanised mouse model. Our study supports the use of available antioxidant drugs, such as NAC, as a general and effective anti-COVID-19 approach.

microbiology↗

bioRxiv2022

Requirement for STAT3 and its target, TFCP2L1 in self-renewal of naïve pluripotent stem cells in vivo and in vitro

We previously demonstrated gradual loss of epiblast during diapause in embryos lacking components of the LIF/IL6 receptor. Here we explore requirement for the downstream signalling transducer and activator of transcription, STAT3 and its target, TFCP2L1, in maintenance of naive pluripotency. Unlike conventional markers, such as NANOG, which remains high in epiblast until implantation, both STAT3 and TFCP2L1 proteins decline during blastocyst expansion, but intensify in the embryonic region after induction of diapause, as observed visually and confirmed using our novel image analysis tool, consistent with our previous transcriptional expression data. Embryos lacking STAT3 or TFCP2L1, underwent catastrophic loss of most of the inner cell mass during the first few days of diapause, implicating involvement of signals in addition to LIF/IL6 for sustaining naive pluripotency in vivo. By blocking MEK/ERK signalling from the morula stage we could derive embryonic stem cells with high efficiency from STAT3 null embryos, but not those lacking TFCP2L1, suggesting a hitherto unknown additional role for this essential STAT3 target in transition from embryo to embryonic stem cells in vitro. Summary StatementInducing diapause in mouse embryos demonstrates that STAT3 and TFCP2L1 are essential for self-renewal of the epiblast, but only TFCP2L1 is required for derivation of embryonic stem cells.

developmental biology↗

bioRxiv2022

Chemical conversion of human conventional Pluripotent Stem Cells to Trophoblast Stem Cells

In human embryos, naive pluripotent cells of the inner cell mass generate epiblast, primitive endoderm and Trophectoderm (TE) lineage, whence trophoblast cells derive. In vitro, naive pluripotent stem cells (PSCs) retain this potential and can generate trophoblast stem cells (TSCs), while conventional PSCs form amnion-like cells and lack the competence to generate TSCs. Transient histone deacetylase and MEK inhibitions with LIF stimulation can be used to chemically reset conventional to naive PSCs. Here we report that chemical resetting induced expression of both naive and TSC markers and of placental imprinted genes. A modified chemical resetting protocol allowed for the fast and efficient conversion of conventional PSCs into TSCs, entailing shutdown of pluripotency genes and full activation of the trophoblast master regulators, without induction of amnion markers. Chemical resetting generates a responsive intermediate state, in which conventional PSCs rapidly acquire competence to form TSCs without the need of stabilisation and expansion in a naive state. The efficiency and rapidity of our system will be useful for the study of cell fate transitions, and to generate models of placental disorders.

developmental biology↗

bioRxiv2022

Distinct phospho-variants of STAT3 regulate naïve pluripotency and developmental pace in vivo

STAT3 has been studied extensively in the context of self-renewal of naive pluripotent mouse embryonic stem cells. We uncovered acute roles for STAT3 and its target, TFCP2L1, in maintenance of epiblast and primitive endoderm during in vivo diapause. On an outbred genetic background, we observed consistent developmental retardation from implantation until embryonic day 11.5, beginning with significant reduction of epiblast cells at implantation in Stat3 null embryos. Remarkably, mutants closely resemble non-affected embryos from the previous day at all postimplantation stages examined. We attribute this phenotype to loss of the active serine phosphorylated form of STAT3 required for neural differentiation and implicated in growth defects in mice and humans. Bulk RNA-sequencing analysis of isolated epiblasts revealed compromised lipid metabolism in Stat3 null embryos by embryonic day 6.5. Furthermore, we demonstrate that gastruloids generated from Stat3 null ESCs failed to extend the posterior axis or maintain BRACHYURY expression and were underrepresented in this region when mixed with wild type cells in chimaeric gastruloids. Our study implicates a role for STAT3 in temporal control of embryonic progression and metabolic mechanisms.

developmental biology↗

bioRxiv2021

STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia

STAT3 and HIF1 are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. Here we report that STAT3 is required for the HIF1-dependent response to hypoxia. In Stat3 knock-out pluripotent embryonic stem cells (ESCs), a large fraction of HIF1 target genes is not induced by hypoxia. Mechanistically, STAT3 does not regulate neither HIF1 expression nor stability, rather, it physically interacts with it in the nucleus. In vivo, we observed that both genetic and chemical inactivation of Stat3 blunted physiological responses to hypoxia, such as angiogenesis, erythropoiesis, and immune cell mobilization. Such defects were accompanied with faulty transcriptional activity of HIF1. In sum, our data reveal that STAT3 and HIF1 cooperatively mediate the physiological response to hypoxia.

developmental biology↗

bioRxiv2021

PsiNorm: a scalable normalization for single-cell RNA-seq data

Single-cell RNA sequencing (scRNA-seq) enables transcriptome-wide gene expression measurements at single-cell resolution providing a comprehensive view of the compositions and dynamics of tissue and organism development. The evolution of scRNA-seq protocols has led to a dramatic increase of cells throughput, exacerbating many of the computational and statistical issues that previously arose for bulk sequencing. In particular, with scRNA-seq data all the analyses steps, including normalization, have become computationally intensive, both in terms of memory usage and computational time. In this perspective, new accurate methods able to scale efficiently are desirable. Here we propose PsiNorm, a between-sample normalization method based on the power-law Pareto distribution parameter estimate. Here we show that the Pareto distribution well resembles scRNA-seq data, independently of sequencing depths and technology. Motivated by this result, we implement PsiNorm, a simple and highly scalable normalization method. We benchmark PsiNorm with other seven methods in terms of cluster identification, concordance and computational resources required. We demonstrate that PsiNorm is among the top performing methods showing a good trade-off between accuracy and scalability. Moreover PsiNorm does not need a reference, a characteristic that makes it useful in supervised classification settings, in which new out-of-sample data need to be normalized. PsiNorm is available as an R package available at https://github.com/MatteoBlla/PsiNorm

bioinformatics↗

bioRxiv2020

Mitochondrial STAT3 regulates proliferation of tissue stem cells.

The STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined in vivo that mitochondrial STAT3 regulates mtDNA transcription in embryonic and larval stem cell niches and that this activity affects their proliferation rates. As a result, we demonstrated that STAT3 import inside mitochondria requires Y705 phosphorylation by Jak2, while its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. These data were confirmed using mouse embryonic stem cells: Y705 mutated STAT3 cannot enter the mitochondrion while the S727 mutation does not affect mitochondrial import of the protein. Surprisingly, STAT3-dependent increase of mitochondrial transcription seems independent from STAT3 binding to STAT3 responsive elements. Finally, loss of function experiments, with chemical inhibition of JAK/STAT3 pathway or genetic ablation of stat3 gene, demonstrated that STAT3 is also required for cell proliferation in the intestine of zebrafish.

developmental biology↗

bioRxiv2020

Embryonic stem cells commit to differentiation by symmetric divisions following a variable lag period

Mouse embryonic stem (ES) cells are derived from the epiblast of the preimplantation embryo and retain the capacity to give rise to all embryo lineages. ES cells can be released into differentiation from a near-homogeneous maintenance condition. Exit from the ES cell state can be accurately monitored using the Rex1-GFPd2 transgenic reporter, providing a powerful system for examining a mammalian cell fate transition. Here, we performed live-cell imaging and tracking of ES cells during entry into differentiation for 48 hours in defined conditions. We observed a greater cell surface area and a modest shortening of the cell cycle prior to exit and subsequently a reduction in cell size and increase in motility. We did not see any instance of cells regaining ES cell identity, consistent with unidirectional developmental progression. Transition occurred asynchronously across the population but genealogical tracking revealed a high correlation in cell-cycle length and Rex1-GFPd2 expression between daughter cells. A population dynamics model was consistent with symmetric divisions during exit from naive pluripotency. Collapse of ES cell identity occurred acutely in individual cells but after a variable delay. The variation in lag period can extend up to three generations, creating marked population asynchrony.

developmental biology↗

bioRxiv2020

Allelic expression analysis of Imprinted and X-linked genes from bulk and single-cell transcriptomes

Genomic imprinting and X chromosome inactivation (XCI) are two prototypical epigenetic mechanisms whereby a set of genes is expressed mono-allelically in order to fine-tune their expression levels. Defects in genomic imprinting have been observed in several neurodevelopmental disorders, in a wide range of tumours and in induced pluripotent stem cells (iPSCs). Single Nucleotide Variants (SNVs) are readily detectable by RNA-sequencing allowing determination of whether imprinted or X-linked genes are aberrantly expressed from both alleles, although standardised analysis methods are still missing. We have developed a tool, named BrewerIX, that provides comprehensive information about the allelic expression of a large, manually-curated set of imprinted and X-linked genes. BrewerIX does not require programming skills, runs on a standard personal computer, and can analyze both bulk and single-cell transcriptomes of human and mouse cells directly from raw sequencing data. BrewerIX confirmed previous observations regarding the bi-allelic expression of some imprinted genes in naive pluripotent cells and extended them to preimplantation embryos. BrewerIX identified also misregulated imprinted genes in breast cancer cells and in human organoids and identified new genes escaping XCI in human somatic cells. We believe BrewerIX will be useful for the study of genomic imprinting and XCI during development and reprogramming, and for detecting aberrations in cancer, iPSCs and organoids. Due to its ease of use to non-computational biologists, its implementation could become standard practice during sample assessment, thus raising robustness and reproducibility of future studies.

bioinformatics↗

bioRxiv2017

A common molecular logic determines embryonic stem cell self-renewal and reprogramming

During differentiation and reprogramming new cell identities are generated by reconfiguration of gene regulatory networks. Here we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naive pluripotency. We find that a Boolean network architecture defined for maintenance of naive state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single cell resolution. Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 136 predictions formulated by the dynamic network, finding a predictive accuracy of 78.7%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naive pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.

developmental biology↗