Biology subjects

Taoudi, S.

Publications and source records attributed to Taoudi, S..

7 recordsLinked to original sources

bioRxiv2024

Clonal analysis of fetal hematopoietic stem/progenitor cell subsets reveals how post-transplantation capabilities are distributed

It has been proposed that adult haematopoiesis is sustained by multipotent progenitor (MPP) clones that are specified during development. From an immunophenotypic perspective, it is known that hematopoietic stem cell (HSC) and MPPs are present in the fetal liver yet our understanding of how fetal MPPs functionally compare to those in the adult bone marrow is incomplete. Using acute-term transplantations, we found that at a population-level fetal immunophenotypic MPP classes exhibited similar lineage biases as adult cells, albeit with some difference in lymphoid output. Clonal assessment of fetal MPPs engraftment revealed that lineage biases largely resulted from differences in the pattern of single-or bi-lineage differentiation. Immunophenotypic long-term (LT)-and short-term (ST)-HSCs in the fetal liver were distinguished from MPPs according to propensity for clonal multi-lineage differentiation. We also discovered that a large cohort of long-term repopulating units (LT-RU) were within the immunophenotypic ST-HSC population, a significant portion of these were labelled using Flt3-cre. This finding has two implications: (1) use of the CD150+ LT-HSC immunophenotype alone will systematically underestimate the size and diversity of the fetal LT-RU pool; and, (2), given fetal LT-RUs with a ST-HSC immunophenotype have the functional attributes required to persist into adulthood.

developmental biology↗

bioRxiv2023

A Risk-reward Examination of Sample Multiplexing Reagents for Single Cell RNA-Seq

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNASeq experiments, facilitating more accurate and cost-effective studies.

genomics↗

bioRxiv2023

LoxCode in vivo clonal barcoding resolves mammalian epiblast contribution to fetal organs

Much remains to be learned about the clonal fate of mammalian epiblast cells in vivo. Here we develop a high diversity, high throughput, Cre recombinase-driven DNA LoxCode barcoding technology for in vivo clonal lineage tracing. E5.5 pre-gastrulation embryos were barcoded in utero and epiblast clones later assessed for their contribution to a wide range of tissues and cell types in the E12.5 organogenesis-stage embryo. While a few epiblast clones contributed broadly to most tissues and cell types of the three germ layers, many clones were lineage biased towards either blood, ectoderm lineages, mesenchymal tissues or limbs. In addition to lineage bias, most epiblast clones were differentially fated for tissue types or descendants in tissues compartments across the body axes. Using a stochastic agent-based model of embryogenesis and LoxCode barcoding, we inferred and experimentally validated predicted cell fate biases as well as clonal compositions across tissues that are consistent with events of lineage segregation and shared trajectory of lineage differentiation. Our study has demonstrated the power of LoxCode barcoding in investigating multi-modal clonal fate at high throughput, thus enabling an in-depth interrogation of the clonal contribution of E5.5 epiblast cells to fetal tissues, organs and body parts.

developmental biology↗

bioRxiv2022

Analysis of organelle content supports a membrane budding model of platelet biogenesis

Understanding how in vivo platelet biogenesis is undertaken is critical to making on-demand platelet production for clinical use feasible. We recently described the discovery of plasma membrane budding as a major in vivo platelet-producing pathway. In vitro recapitulation of this finding could pave the way towards efficient laboratory-based platelet production. The plausibility of the plasma membrane budding model has been called into question. The foundation of this is the contention that the size and payload composition of plasma membrane buds are not consistent with bona fide platelets. Thus, membrane buds likely represent stages in megakaryocyte-derived microparticle formation. Using 3D super-resolution imaging, we have performed a quantitative comparison of size and organelle content of plasma membrane buds, platelets, and microparticles in the adult mouse bone marrow. We unequivocally demonstrate that the structures we previously described as membrane buds exhibit the same size range as free platelets, that all buds contain organelles, and that membrane buds and free platelets contained an equivalent number of organelles. Crucially, membrane buds and microparticles are completely distinct from each other. To prevent future confusion between the processes of microparticle formation and platelet biogenesis, we propose using the more specific term "pre-platelet membrane buds".

cell biology↗

bioRxiv2021

Mesoderm-Derived PDGFRA+ Cells Regulate the Emergence of Hematopoietic Stem Cells in the Dorsal Aorta

Mouse hematopoietic stem cells (HSCs) first emerge at embryonic day 10.5 (E10.5) on the ventral surface of the dorsal aorta, by endothelial-to-hematopoietic transition (EHT). We investigated whether cells with mesenchymal stem cell-like activity, which provide an essential niche for long-term HSCs (LT-HSCs) in the bone marrow, reside in the aorta- gonad-mesonephros (AGM) and contribute to the structural development of the dorsal aorta and EHT. Using transgenic mice, we demonstrate a lineage hierarchy for AGM stromal cells and traced the E10.5/E11.5 aortic endothelium and HSCs to mesoderm derived (Mesp1) PDGFRA+ stromal cells (Mesp1der PSCs). Mesp1der PSCs dominate the sub-endothelial and ventral stroma in the E10.5-E11.5 AGM but by E13.5 were replaced by neural crest (Wnt1) derived PDGFRA+ stromal cells (Wnt1der PSCs). Co-aggregating non-hemogenic embryonic and adult endothelial cells with Mesp1der PSCs but not with Wnt1der PSCs resulted in activation of a hematopoietic transcriptional program in endothelial cells accompanied by EHT and generation of LT-HSCs. Dose-dependent inhibition of PDGFRA signalling or BMP, WNT, NOTCH signalling interrupted this reprogramming event. This partnership between endothelial cells and AGM Mesp1der PSCs could potentially be harnessed to manufacture LT-HSCs from endothelium.

developmental biology↗

bioRxiv2020

Quantitation analysis by flow cytometry shows that Wt1 is required for development of the proepicardium and epicardium

The epicardium is a cell layer found on the external surface of the heart. During development it has an epithelial identity and contains progenitor cells for coronary smooth muscle and cardiac fibroblasts. The epicardium has been suggested to have therapeutic potential in cardiac repair. Study of epicardial development has been difficult because it is dynamic and morphologically complex. We developed a flow cytometry-based method to quantify cardiac development including the epicardial lineage. This provided accurate and sensitive analysis of (1) the emergence of epicardial progenitors within the proepicardium (2) their transfer to the heart to form the epicardium, and (3) their epithelial-to-mesenchymal transition (EMT) to create the subepicardium. Platelet-derived growth factor alpha (Pdgfra) and Wilms tumor protein (Wt1) have both been reported to be pro-mesenchymal during epicardial EMT. Quantitative analysis with flow cytometry confirmed a pro-mesenchymal role for Pdgfra but not for Wt1. Analysis of Wt1 null embryos showed that they had (1) poor formation of proepicardial villi, (2) reduced transfer of proepicardial cells to the heart, (3) a discontinuous epicardium with poor epithelial identity, and (4) a proportionally excessive number of mesenchymal-like cells. This data shows that Wt1 is essential for epicardial formation and maintenance rather than being pro-mesenchymal.

developmental biology↗

bioRxiv2020

The clonal and molecular aetiology of emergency dendritic cell development

Extrinsic regulation of single haematopoietic stem and progenitor cell (HSPC) fate is crucial for immune cell development. Here, we examine the aetiology of Flt3 ligand (Flt3L)-mediated emergency development of type 1 conventional dendritic cells (cDC1s), which results in enhanced immunity against infections and cancer. Using cellular barcoding, we demonstrate a predominant role of enhanced clonal expansion and moderate contribution via recruitment of additional cDC1-generating HSPCs. The selective cDC1 expansion occurs primarily via multi-/oligo-potent clones, without compromising output to other lineages. To understand the molecular hallmarks early during a Flt3L response, we develop Divi-Seq to simultaneously profile cell division history, surface phenotype and transcriptional state of single HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative early progenitor-like state, which leads to selective emergence of CD11c+cKit+ transitional precursors with high cellular output to cDC1s. These findings inform the mechanistic action of Flt3L in natural immunity and immunotherapy at a clonal level.

immunology↗