In vitro fertilisation | EPFL Graph Search

In vitro fertilisation (IVF) is a process of fertilisation where an egg is combined with sperm in vitro ("in glass"). The process involves monitoring and stimulating a woman's ovulatory process, removing an ovum or ova (egg or eggs) from her ovaries and letting sperm fertilise them in a culture medium in a laboratory. After the fertilised egg (zygote) undergoes embryo culture for 2–6 days, it is transferred by catheter into the uterus, with the intention of establishing a successful pregnancy. IVF is a type of assisted reproductive technology used for infertility treatment, gestational surrogacy, and, in combination with pre-implantation genetic testing, avoiding transmission of genetic conditions. A fertilised egg from a donor may implant into a surrogate's uterus, and the resulting child is genetically unrelated to the surrogate. Some countries have banned or otherwise regulate the availability of IVF treatment, giving rise to fertility tourism. Restrictions on the availability o

Isolation and characterisation of a porcine cardiac adherent cell population from heart biopsies

Christelle Hasselmann

Cardiac diseases represent the major cause of mortality in the Western world. The discovery of the promising human Cardiac Adherent Proliferating (CAP) cells for a potential cardiac cell therapy by the Tissue Engineering group induced the need of a reliable animal model. The aim of the project is to identify a porcine cardiac cell population having similar characteristics as human CAP cells. Here a protocol for isolating cells from porcine heart biopsies have been established, as well as the in vitro expansion method. Additionally, the characterization of those expanded porcine cardiac cells was accomplished by growth kinetics study, FACS analysis, multilineage potential and myogenic induction assays. In this study we identified a not yet described porcine cardiac adherent cell population directly isolated from biopsies within the inner right ventricle. With almost

5\times {10}^{7}

expanded cells after 30 days in culture, porcine cardiac cells present a high rate of proliferation in vitro. Porcine cardiac cells maintain a swirl-like pattern until 5-6 days in culture, and become fibroblast-like with a parallel organization when confluent. FACS analysis indicated that porcine cells are mainly CD90+ in opposite to human CAP cells. Both species show a similar CD45- and CD44+ profile. The multilineage assay showed no histological differentiation into fat, bone and cartilage on porcine cardiac cells like on human CAP cells. However gene expression analysis on porcine cardiac cells indicated that aP2 was upregulated in population undergoing an adipogenic induction, as well as osteopontine expression in osteogenic induction population. Porcine cardiac cell myogenic induction potential was tested on C2C12 myogenic mouse cells. The mouse cells differentiated in muscle in vitro thanks to the influence of those cardiac cells. Both cell populations have similar characteristics and the main distinction is the different CD90 expression profile. Those results encourage to realise additional studies, to confirm that those isolated cells could be a reliable model of human CAP cells.

2009

Expansion of a VE-Cadherin+CD45+ population in the yolk sac upon Etv2 overexpression in the mouse embryo - Effects of BMP and Wnt signaling inhibition on the differentiation of a Flk1+ extraembryonic mesoderm in vitro model

Matteo Pluchinotta

The difficulty to find compatible donors for bone marrow transplantation makes the need for an alternative source of HSCs urgent. HSC derived from patient-specific iPS cells are ideal candidates for this purpose. Nevertheless, although HSCs are the best characterized adult stem cells, the in vitro generation and expansion of bone fide HSCs still has not been achieved without transgene insertion. Successful derivation of HSCs from ES cells or iPS cells will require a comprehensive understanding of inductive signals and downstream effectors involved in the normal arising of HSCs during embryonic development. BMP and Wnt signaling pathways play a very important role in this differentiation process. Here, we report how BMP and Wnt signaling inhibition cooperatively alter the differentiation of Flk1+PDGFRα- cells after reculture in serum-free, feeder layer-free conditions. This data will be useful in determining the factors that have to be added to a chemically defined culture medium aimed at derivation of bone fide HSC in vitro. In the second part of this master’s project, we studied the spatial and temporal localization requirements of Etv2 expression, a key regulator of the earliest events in the development of the hematopoietic and vascular systems. To gain more insight on this matter, we conditionally knocked-in Etv2 in the early endothelial compartment, using a Tie2Cre system. This leaded to an embryonically lethal phenotype by E11.5, and most interestingly, to the expansion of a VE-Cadherin+CD45+ population in the E10.5 yolk sac. We believe that these cells represent a increased pool of immature pre-definitive hematopoietic stem cells, thus underlining further the importance of Etv2 in the differentiation process towards hematopoietic, and endothelial lineages in the mouse embryo.

2011

Immunosuppressive roles of Activin-A in melanoma and characterization of its precursor cleavage

Katarina Pinjusic

Activin-A, a transforming growth factor êµ family member, is a pleiotropic cytokine with diverse functions in development, fertility, adult tissue homeostasis, and aging. Accordingly, deregulation of Activin-A signaling has been associated with many pathological conditions, including cancer and cancer-associated muscle wasting (cachexia). Activin-A was shown to signal in endocrine, but also local autocrine and paracrine manner. Its secretion in many cancer types is associated with a poor prognosis. Previous studies suggest that Activin-A expression in melanoma promotes tumor growth and metastasis indirectly by inhibiting adaptive anti-tumor immunity, leading to a decrease in the intratumoral frequency of cytotoxic CD8+ T cells (CTLs). However, the underlying mechanisms and the relevant direct target cells of Activin-A and their significance for cancer therapies remained unknown. Similar to other TGFêµ-related ligands, proActivin-A is cleaved by proprotein convertases (PCs) to release mature ligands. However, unlike other family members whose signaling is tightly regulated, Activin-A apparently does not have latency.In this thesis, I characterize proActivin-A cleavage and show that one prodomain can be cleaved independently of known PCs. The resulting hemi-cleaved Activin-A can bind activin type II receptors and induce SMAD3 signaling in reporter cells. A protein interactome screen and analysis of cleavage intermediates in plasma revealed that the hemi-cleaved Activin-A likely interacts with extracellular matrix proteins via the remaining prodomain where it promotes local signaling and regulates trafficking. Luciferase reporter assay showed that hemi-cleaved Activin-A can avoid, at least partially, inhibition by endogenous inhibitors FSTL3 and FST, and enable parallel activation of BMP and Activin signaling pathways. Finally, analysis of B16-F1 melanoma models revealed that only fully cleaved Activin-A in source cells increased tumor growth by acting locally in the tumor microenvironment suggesting that the context of Activin-A precursor processing regulates its tumor growth-promoting effect.The second part of this thesis addresses the mechanisms of Activin-induced immunosuppression in melanoma. We found that Activin-A impairs CTL function indirectly and independently of CD4+ T cell, CSF1R+ macrophages, and IL4 signaling. At least in part, anti-tumor CTL responses are impaired by the inhibitory effects of Activin-A on the secretion of critical CTL-recruiting chemokines CXCL9 and CXCL10 by macrophages and dendritic cells (DCs). Furthermore, Activin-A conferred resistance to immune checkpoint blockade therapy. Characterization of Activin-induced changes in the gene expression of tumor-infiltrating cells by Single cell RNA sequencing revealed that INHBA expression induced the most changes in DCs and least in T cells. Interestingly, the most prominently induced hallmark in Activin-secreting tumors across cell types was the IFN signature. In vitro analyses showed that Activin-A increases IFNÎ³-induced activation of STAT1 and confers resistance to cytostatic IFN signaling in cancer cells.Altogether, the findings of this thesis expand our knowledge on the regulation of Activin-A signaling by precursor cleavage and offer insights into mechanisms of Activin-induced immunosuppression in melanoma. Inhibition of Activin-A maturation thus emerges as a potential new strategy to improve responses to immunotherapies in melanoma and likely other cancers

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