Preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD or PIGD) is the genetic profiling of embryos prior to implantation (as a form of embryo profiling), and sometimes even of oocytes prior to fertilization. PGD is considered in a similar fashion to prenatal diagnosis. When used to screen for a specific genetic disease, its main advantage is that it avoids selective abortion, as the method makes it highly likely that the baby will be free of the disease under consideration. PGD thus is an adjunct to assisted reproductive technology, and requires in vitro fertilization (IVF) to obtain oocytes or embryos for evaluation. Embryos are generally obtained through blastomere or blastocyst biopsy. The latter technique has proved to be less deleterious for the embryo, therefore it is advisable to perform the biopsy around day 5 or 6 of development. The world's first PGD was performed by Handyside, Kontogianni and Winston at the Hammersmith Hospital in London. Female embryos were selectively transferred in five couples at risk of X-linked disease, resulting in two twins and one singleton pregnancy. The term preimplantation genetic screening (PGS) refers to the set of techniques for testing whether embryos (obtained through IVF/ICSI) have abnormal chromosomes' number. In other words, it tests if an embryo is aneuploid or not. PGS is also called aneuploidy screening. PGS was renamed preimplantation genetic diagnosis for aneuploidy (PGD-A) by Preimplantation Genetic Diagnosis International Society (PGDIS) in 2016. The PGD allows studying the DNA of eggs or embryos to select those that carry certain mutations for genetic diseases. It is useful when there are previous chromosomal or genetic disorders in the family and within the context of in vitro fertilization programs. This kind of tests are need because hereditary disorders are related with 20% of child deaths in developed countries . It is estimated that , as a whole , they are responsible for around 18% of pediatric hospital admissions.

In vitro modeling of early mouse development

Mehmet Ugur Girgin

Previous attempts to recapitulate embryogenesis in a developmentally relevant context started with aggregates composed of a few thousand ESCs, termed embryoid bodies (EB), that upon induction of differentiation reveal a surprising level of autonomous cell fate patterning, in some cases giving rise to spatially arranged ectoderm, mesoderm and endoderm layers. However, cell fate patterning and morphogenesis in EBs is poorly reproducible and cannot be controlled. These limitations necessitate further research on the self-organization potential of ESCs, and in particular the development of more robust in vitro models mimicking post-implantation mouse embryos. This PhD thesis explored three novel approaches towards this goal. Firstly, together with collaborators, I focused on a previously established in vitro model of gastrulation termed gastruloids. Gastruloids emerge from small ESC aggregates that, upon activation of Wnt signaling, break symmetry and undergo axial morphogenesis to create structures that are similar to the developing tail of the mouse embryo. A systematic improvement of the existing culture conditions allowed gastruloids to be cultured for an extended period of time, resulting in an embryo-like patterning along the three major body axes. Furthermore, gene expression analyses, combined with immunohistochemistry and in situ hybridization assays, demonstrated that gastruloids exhibit spatio-temporal activation of Hox genes in a way that is remarkably similar to post-implantation mouse embryos. Secondly, I report a novel gastruloid culture approach, which for the first time promotes the formation of anterior neural tissues. Mouse ESCs were aggregated and treated in high-throughput in a bioengineered microwell array system to generate homogeneous âartificial epiblastsâ that transcriptionally and morphologically resemble post-implantation epiblasts. These epiblast-like (EPI) aggregates break symmetry and undergo axial elongation to establish antero-posterior patterning. I provide evidence for attenuated Wnt signaling and the presence of an epithelium in the starting EPI aggregates as the major determinants for the formation of anterior neural tissues, that are completely absent in conventional gastruloids. Lastly, I systematically explored the role of extraembryonic tissues in the patterning of artificial epiblasts. By combining EPI aggregates with trophoblast stem cell aggregates, hybrid self-organizing structures termed EpiTS embryoids were generated that mimic epiblast and extraembryonic ectoderm, respectively. EpiTS embryoids were found to execute an elaborate self-organization program that is initiated through highly localized gastrulation-like processes at the embryonic-extraembryonic interface. Upon axial elongation, EpiTS embryoids give rise to primitive brain-like and preplacodal ectoderm-like regions. These observations highlight the role of extraembryonic tissues for proper induction of gastrulation and in particular the formation of anterior neural tissues. Altogether, this thesis introduces three innovative and highly versatile stem cell culture technologies to study post-implantation mouse embryonic development. The approaches are amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing experimental tools. This work should contribute to the advancement of the nascent field of synthetic embryology.

EPFL2020

Exploring the potency of normal and pathological human thymic epithelial cells

Tiphaine Mélodie Chantal Arlabosse

Thymic epithelial cells (TECs) are organised in a unique 3D network that is critical for the development of efficient and self-tolerant T-cells. We report for the first time that the human thymus of all ages, even old and involuted, contains a significant number (up to 30%) of clonogenic TECs with extensive growth potential. All clonogenic TECs derive from a common EpCAM+ precursor, which within 48h in culture undergoes a molecular switch and gives rise to refringent EpCAM- colonies and stratified EpCAM+ colonies. The latter two subpopulations can be distinguished based on their morphology, their expression of genes implicated in epidermal stratification and their expression of genes implicated in epithelia-mesenchymal transition. Clonal analysis revealed that clonogenic cells are hierarchically organised, refringent cells being higher in the hierarchy than stratified cells. Although the thymus normally involutes with age, it can be the site of uncontrolled proliferation, resulting in the formation of thymic tumours including thymomas. Thymomas are classified by the WHO based on histological criteria as type A, B and AB and are frequently associated with Myasthenia Gravis, an autoimmune disease affecting the neuromuscular junction. The biology of thymomas is poorly understood, because of the rarity of the tumours and because of the lack of a reliable in vitro model. We report for the first time, that clonogenic cells can be isolated from all thymoma subtypes as well as from thymic hyperplasia and mediastinal teratoma. The latter cells displayed many similarities with normal TECs but also differences in terms of morphology, gene expression and genetic profile. Importantly, all thymic tumours studied contained refringent-like EpCAM- cells and stratified EpCAM+ cells, supporting the hypothesis that thymic tumour-initiating cells arise from clonogenic TECs. We have not succeeded yet in developing an appropriate assay to support the growth of clonogenic epithelial cells from thymic tumours in vivo. Finally, we report that the SY5 monoclonal antibody raised against human involucrin reproducibly cross-reacts with a molecule present in the Golgi apparatus of refringent cells derived from normal and pathological thymi. Immunocytochemistry and knock-down experiments revealed that the latter molecule is not involucrin. Epitope mapping by phage-display experiments and data base search identified putative candidate proteins, including nesprin-1, a player of the neuromuscular junction, which could be the key to understand the frequent association of thymomas with Myasthenia Gravis.

EPFL2017

Studies on lentiviral-mediated transgenesis

Marc-Olivier Sauvain

Transgenic animals are essential research tools, whether to address basic biological questions or to develop preclinical models of human diseases. Their generation through the injection of naked plasmid DNA into the male pronucleus of a fertilized oocyte has been the standard practice for almost 3 decades. However, this approach is invasive, largely limited to the mouse and results in low rates of transgene integration. Although the inefficiency of pronuclear injection can be overcome in small animals by high-throughput screening for transgene integration, this becomes economically more challenging in larger animals, such as sheep, pigs or cows, because of the extreme costs associated with this procedure (

60'000-

300'000). Thus, new strategies to enhance the production and the variety of transgenic animals would be an important asset. One such approach has emerged through the use of lentiviral vectors, a gene delivery system that can efficiently transfer and stably integrate its cargo into a wide variety of cell types from numerous species, and has been successfully applied to generate transgenic mice, rats, pigs and cows. The increasing use of lentivector-mediated transgenesis warrants a full analysis of its modalities. As a step towards this goal, the present study aimed at characterizing the genotypic features of transgenic mice generated by lentiviral infection of zygotes. First, as an indirect method to measure the kinetics of lentiviral integration in early embryos, we examined the rates of transmission of individual proviruses from G0 transgenic mice to their G1 progeny using a PCR-based technique specific for each integrant. The mean rate of transmission of 44% for individual proviruses suggests that vector integration was most often established between the post-S phase of the one-cell stage and pre-S phase of the two-cell stage. We then moved on to define proviral integration site selection in lentivector-generated transgenic mice. Using LAM-PCR-mediated amplification and sequencing of the host genome-provirus junctions, we found that the frequency of proviral integration inside a gene was 1.75- and 1.88-fold higher in transgenic mice and 3T3 fibroblasts, respectively, than expected from the distribution of annotated genes in the mouse genome (Pmice .03e 09; P3T3 5.55e 16). Moreover, integration was not influenced by the transcriptional orientation of the target gene and tended to favour the middle of transcribed regions. We also observed a subtle difference in the integration patterns of lentiviral vectors in 3T3 fibroblasts and transgenic mice, with the latter exhibiting a slightly higher frequency of integration into repeats. Although this difference was statistically not significant, it might reflect the elevated transcriptional activity of repetitive elements in preimplantation embryos. Since proviruses favour intragenic localization, we analyzed G2 mice homozygous for specific lentiviral integrants using PCR-based techniques and observed that the frequency of homozygosity was below the expected mendelian transmission rate of 25%. This suggested that some of the homozygous G2 mice must be nonviable due to the potential of retrovirus integrants to inactivate target genes. However, the normal rates of G1 representation of proviruses that did not achieve homozygosity in G2 suggested an absence of toxicity in the heterozygous state. Processes governing the earliest steps of mammalian development are still incompletely understood. In particular, the stage at and modalities by which early blastomeres become committed to either the inner cell mass (ICM), which subsequently yields the embryo proper, or the trophectoderm, which serves as precursor of extra-embryonic tissues, is unclear. The long held "random" hypothesis for development of the embryonic-abembryonic axis states that, within these two structures, early blastomeres are equally represented. However, recent lineage-tracing experiments support a model of "developmental bias", in which the progeny of the early blastomeres is allocated in a biased fashion. In our study, we took advantage of the specific integration pattern of lentiviral vector – i.e. each lentiviral vector integrates the host genome at specific sites – to "fingerprint" individual blastomeres and to follow them during embryogenesis. For this, we injected lentiviral vectors into embryos at either the one-cell or the two-cell stage. The Southern Blot (SB) analysis performed at E12.5 revealed that the infection at the one-cell stage yielded integrants shared for their majority by both embryonic and extra-embryonic tissues (74% of similar bands), while the infection at the two-cell stage induced a drastically different picture, with only a minority of common integrants between both types of tissues (22% of similar bands). This suggests that early blastomeres – from the two-cell stage on – contribute differentially to these lineages, which argues against a purely stochastic model. However, we still obtained 22% of shared integrations after infection at the two-cell stage, with only 8 out of 27 embryos showing a complete segregation of the proviral integration patterns. This rules out a "fully determined" model. Thus, our observations rather support the "developmental bias" hypothesis.

EPFL2009