Apprentissage profond appliqué à la classification d’images microscopiques embryonnaires

National audienceDans le domaine de la fécondation in vitro (FIV), l'imagerie par time-lapse (ITL) est une technologie qui produit une vidéo montrant le développement de l'embryon pendant ses premiers jours. L'ITL permet d'annoter les instants auxquels commencent et finissent les différentes phases de développement de l'embryon. Cependant, le processus d'annotation prend du temps et nécessite des experts. Dans cet article, nous montrons que pour prédire les phases visibles dans les images issues de vidéos ITL, un ResNet-3D est meilleur qu'un équivalent 2D ou un ResNet-LSTM

Towards deep learning-powered IVF: A large public benchmark for morphokinetic parameter prediction

An important limitation to the development of Artificial Intelligence (AI)-based solutions for In Vitro Fertilization (IVF) is the absence of a public reference benchmark to train and evaluate deep learning (DL) models. In this work, we describe a fully annotated dataset of 704 videos of developing embryos, for a total of 337k images. We applied ResNet, LSTM, and ResNet-3D architectures to our dataset and demonstrate that they overperform algorithmic approaches to automatically annotate stage development phases. Altogether, we propose the first public benchmark that will allow the community to evaluate morphokinetic models. This is the first step towards deep learning-powered IVF. Of note, we propose highly detailed annotations with 16 different development phases, including early cell division phases, but also late cell divisions, phases after morulation, and very early phases, which have never been used before. We postulate that this original approach will help improve the overall performance of deep learning approaches on time-lapse videos of embryo development, ultimately benefiting infertile patients with improved clinical success rates (Code and data are available at https://gitlab.univ-nantes.fr/E144069X/bench_mk_pred.git)

Pannexin-1 Channels govern the Generation of Necroptotic small Extracellular Vesicles

The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) controls the execution of necroptosis, a regulated form of necrosis that occurs in apoptosis-deficient conditions. Active oligomerized MLKL triggers the exposure of phosphatidylserine residues on the cell surface and disrupts the plasma membrane integrity by forming lytic pores. MLKL also governs the biogenesis and shedding of proinflammatory small extracellular vesicles (EVs) during the early steps of necroptosis, however the molecular basis is unknown. Here, we find that MLKL oligomers activate plasma membrane Pannexin-1 (PANX1) channels, concomitantly to the loss of phosphatidylserine asymmetry. This plasma membrane "leakiness" requires the Rab GTPase Rab27 isoforms, which usher the small EVs to their release. Conversely, PANX1 knockdown disorganized the small EVs machinery and precludes vesicles extrusion. These data identify a novel signaling nexus between MLKL, Rab27, and PANX1, and propose ways to interfere with small EV generation

Neuropilin-1 modulates the 3D invasive properties of glioblastoma stem-like cells

International audienceGlioblastoma multiforme (GBM) is a rare, yet devastating, primary brain tumor in adults. Current treatments remain generally ineffective and GBM almost invariably recurs, resulting in median survival of 15 months. This high malignancy sources notably from the resilience and invasive capabilities of tumor cells. Within GBM, exists a population of self-sustaining transformed cells with stem-like properties (GSCs), which are thought to be responsible for tumor initiation, growth, and invasion, as well as recurrence. In the tumor microenvironment, GSCs might be found in the vicinity of brain endothelial cells, which provide a protective habitat. Likewise, these resistant, quiescent GSCs may accumulate in hypoxic zones, away from the perivascular niche, or travel towards the healthy brain parenchyma, by eminently co-opting neuro-vascular tracks. Herein, we established an ex vivo model to explore GSC invasive behavior. We found that patient-derived cells massively invade the collagen matrix. In addition, we described that the glycoprotein Neuropilin-1 (NRP1) contributes to GSC spreading and invasion. Indeed, both RNA interference-mediated silencing and CRISPR-mediated gene editing deletion of NRP1 strongly impaired the 3D invasive properties of patient-derived GSCs and their close localization to the brain blood vessels. Of note, other typical features of GSCs, such as expansion and self-renewal were maintained. From a mechanistic standpoint, this biological effect might rely on the expression of the β3 subunit integrin cell-extracellular matrix adhesive receptor. Our data, therefore, propose a reliable approach to explore invasive properties of patient glioma cells ex vivo and identify NRP1 as a mediator in this malignant process

Detection of all adult Tau isoforms in a 3D culture model of iPSC-derived neurons

Tauopathies are a class of neurodegenerative diseases characterized by the presence of pathological intracellular deposits of Tau proteins. Six isoforms of Tau are expressed in the adult human brain, resulting from alternative splicing of the MAPT gene. Tau splicing is developmentally regulated such that only the smallest Tau isoform is expressed in fetal brain, contrary to the adult brain showing the expression of all 6 isoforms. Induced Pluripotent Stem Cell (iPSC) technology has opened up new perspectives in human disease modeling, including tauopathies. However, a major challenge to in vitro recapitulation of Tau pathology in iPSC-derived neurons is their relative immaturity. In this study, we examined the switch in Tau splicing from fetal-only to all adult Tau isoforms during the differentiation of iPSC-derived neurons in a new 3D culture system. First, we showed that iPSC-induced neurons inside Matrigel-coated alginate capsules were able to differentiate into cortical neurons. Then, using a new assay that allowed both the qualitative and the quantitative analysis of all adult MAPT mRNA isoforms individually, we demonstrated that BrainPhys-maintained neurons expressed the 6 adult MAPT mRNA transcripts from 25 weeks of maturation, making this model highly suitable for modeling Tau pathology and therapeutic purposes

Label-free imaging of large samples: 3D rendering and morphological analysis within histological workflows using serial block face imaging

Abstract Serial block face imaging (SFBI) is a method used to generate 3-dimensional (3D) reconstruction of a sample via serial image acquisition. Several SBFI approaches have been proposed for large samples, differing in the ability to generate contrast as well as in the nature of the detected signal. We propose a new system that detects the endogenous autofluorescence signal of paraffin-embedded samples. The sample preparation is simplified compared to other approaches, and adapted to be integrated into a routine histological preparation. More specifically, it was designed to limit reagent toxicity and to be compatible with downstream histological processing. We show the usefulness of the technique with a wide range of tissues based on the intrinsic autofluorescence signal. Optimization of quality section recovery offers the possibility to develop correlative approaches and multimodal analysis between the 3D dataset with the 2-dimensional (2D) sections. In addition, contrast and resolution of block-face images allow us to successfully perform post processing analysis and morphology quantifications. Overall, our methodology offers a simple, cost effective and rapid approach to obtain quantitative data on a large sample with no specific staining

Pannexin‐1 limits the production of proinflammatory cytokines during necroptosis

International audienceThe activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) controls the execution of necroptosis, a regulated form of necrosis that occurs in apopto-sis-deficient conditions. Active oligomerized MLKL triggers the exposure of phosphatidylserine residues on the cell surface and disrupts the plasma membrane integrity by forming lytic pores. MLKL also governs endosomal trafficking and biogenesis of small extracellular vesicles as well as the production of proin-flammatory cytokines during the early steps of necroptosis; however, the molecular basis continues to be elucidated. Here, we find that MLKL oligomers activate Pannexin-1 (PANX1) channels , concomitantly to the loss of phosphatidylserine asymmetry. This plasma membrane "leakiness" requires the small GTPase RAB27A and RAB27B isoforms, which regulate intracellu-lar vesicle trafficking, docking, and fusion with the plasma membrane. Although cells in which PANX1 is silenced or inhibited normally undergo necroptotic death, they display enhanced production of cytokines such as interleukin-8, indicating that PANX1 may tamper with inflammation. These data identify a novel signaling nexus between MLKL, RAB27, and PANX1 and propose ways to interfere with inflammation associated with necroptosis

PiT2 deficiency prevents increase of bone marrow adipose tissue during skeletal maturation but not in OVX-induced osteoporosis

International audienceThe common cellular origin between bone marrow adipocytes (BMAds) and osteoblasts contributes to the intimate link between bone marrow adipose tissue (BMAT) and skeletal health. An imbalance between the differentiation ability of BMSCs towards one of the two lineages occurs in conditions like aging or osteoporosis, where bone mass is decreased. Recently, we showed that the sodium-phosphate co-transporter PiT2/SLC20A2 is an important determinant for bone mineralization, strength and quality. Since bone mass is reduced in homozygous mutant mice, we investigated in this study whether the BMAT was also affected in PiT2 -/- mice by assessing the effect of the absence of PiT2 on BMAT volume between 3 and 16 weeks, as well as in an ovariectomy-induced bone loss model. Here we show that the absence of PiT2 in juveniles leads to an increase in the BMAT that does not originate from an increased adipogenic differentiation of bone marrow stromal cells. We show that although PiT2 -/- mice have higher BMAT volume than control PiT2 +/+ mice at 3 weeks of age, BMAT volume do not increase from 3 to 16 weeks of age, leading to a lower BMAT volume in 16-week-old PiT2 -/- compared to PiT2 +/+ mice. In contrast, the absence of PiT2 does not prevent the increase in BMAT volume in a model of ovariectomy-induced bone loss. Our data identify SLC20a2/PiT2 as a novel gene essential for the maintenance of the BMAd pool in adult mice, involving mechanisms of action that remain to be elucidated, but which appear to be independent of the balance between osteoblastic and adipogenic differentiation of BMSCs

Table_1_PiT2 deficiency prevents increase of bone marrow adipose tissue during skeletal maturation but not in OVX-induced osteoporosis.docx

The common cellular origin between bone marrow adipocytes (BMAds) and osteoblasts contributes to the intimate link between bone marrow adipose tissue (BMAT) and skeletal health. An imbalance between the differentiation ability of BMSCs towards one of the two lineages occurs in conditions like aging or osteoporosis, where bone mass is decreased. Recently, we showed that the sodium-phosphate co-transporter PiT2/SLC20A2 is an important determinant for bone mineralization, strength and quality. Since bone mass is reduced in homozygous mutant mice, we investigated in this study whether the BMAT was also affected in PiT2-/- mice by assessing the effect of the absence of PiT2 on BMAT volume between 3 and 16 weeks, as well as in an ovariectomy-induced bone loss model. Here we show that the absence of PiT2 in juveniles leads to an increase in the BMAT that does not originate from an increased adipogenic differentiation of bone marrow stromal cells. We show that although PiT2-/- mice have higher BMAT volume than control PiT2+/+ mice at 3 weeks of age, BMAT volume do not increase from 3 to 16 weeks of age, leading to a lower BMAT volume in 16-week-old PiT2-/- compared to PiT2+/+ mice. In contrast, the absence of PiT2 does not prevent the increase in BMAT volume in a model of ovariectomy-induced bone loss. Our data identify SLC20a2/PiT2 as a novel gene essential for the maintenance of the BMAd pool in adult mice, involving mechanisms of action that remain to be elucidated, but which appear to be independent of the balance between osteoblastic and adipogenic differentiation of BMSCs.</p

Image_2_PiT2 deficiency prevents increase of bone marrow adipose tissue during skeletal maturation but not in OVX-induced osteoporosis.tif

The common cellular origin between bone marrow adipocytes (BMAds) and osteoblasts contributes to the intimate link between bone marrow adipose tissue (BMAT) and skeletal health. An imbalance between the differentiation ability of BMSCs towards one of the two lineages occurs in conditions like aging or osteoporosis, where bone mass is decreased. Recently, we showed that the sodium-phosphate co-transporter PiT2/SLC20A2 is an important determinant for bone mineralization, strength and quality. Since bone mass is reduced in homozygous mutant mice, we investigated in this study whether the BMAT was also affected in PiT2-/- mice by assessing the effect of the absence of PiT2 on BMAT volume between 3 and 16 weeks, as well as in an ovariectomy-induced bone loss model. Here we show that the absence of PiT2 in juveniles leads to an increase in the BMAT that does not originate from an increased adipogenic differentiation of bone marrow stromal cells. We show that although PiT2-/- mice have higher BMAT volume than control PiT2+/+ mice at 3 weeks of age, BMAT volume do not increase from 3 to 16 weeks of age, leading to a lower BMAT volume in 16-week-old PiT2-/- compared to PiT2+/+ mice. In contrast, the absence of PiT2 does not prevent the increase in BMAT volume in a model of ovariectomy-induced bone loss. Our data identify SLC20a2/PiT2 as a novel gene essential for the maintenance of the BMAd pool in adult mice, involving mechanisms of action that remain to be elucidated, but which appear to be independent of the balance between osteoblastic and adipogenic differentiation of BMSCs.</p