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Trophoblast stem cells
Journal of Reproductive Immunology 115 (2016) 39–55
Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Abstracts of invited and selected oral presentations
Opening lecture S1 Gene editing – From modeling diseases to treating patients T. Cathomen Medical Center – University of Freiburg, Germany Targeted genome editing with designer nucleases has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional “gene addition” type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise “gene surgery”. Moreover, designer nuclease technology allows for novel genetic interventions to fight infectious diseases, including chronic viral infections. We have developed highly specific designer nucleases based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. In preclinical models we demonstrate that TALENs and CRISPR-Cas9 nucleases can be employed to generate cells resistant to infection with human immunodeficiency virus type 1 (HIV-1) or to correct primary immunodeficiencies. Strategies to translate these findings into the clinic will be discussed. http://dx.doi.org/10.1016/j.jri.2016.04.117
Session 1: Models to understand the pathogenesis of pregnancy pathologies: from stem cells to animals S2 Trophoblast stem cells C. Kubaczka, H. Schorle ∗ Department of Developmental Pathology, Bonn University Medical School, Bonn, Germany Trophoblast stem cells (TSCs) are derived from polar trophectoderm (TE) or ectoplacental cone and represent the stem cell population of the extraembryonic lineage. TSCs have the ability to self-renew and give rise to all derivatives of the trophectoderm lineage. In recent years, we have begun to understand the molecular network governing TE and TSC-fate. In particular, the factors Tead4, Eomes, Cdx2, Tfap2c, Gata3 and Elf5 are thought to be crucial for 0165-0378/
TE/TSC-fate. Expressed in TE/TSCs, these genes cooperate to repress the somatic pluripotency program leading to specification, maintenance and fixation of the extraembryonic lineage in the early embryo. In an attempt to standardize the TSC-culture, we succeeded to develop a chemically defined media demonstrating, that the cytokines Fgf4 (plus Heparin) and TGF- are sufficient to keep TSC cultures in an undifferentiated state when grown on Matrigel coated dishes. Further, we were able to demonstrate, that overexpression of four factors (Tfap2c, Eomes, Gata3 and Ets2) suffices to reprogram murine fibroblasts to bona fide induced-TSC (iTSC). The iTSC were fully functional with almost identical expression profile and epigenetic signature when compared to TSC. These data provide the scaffold for a more detailed understanding of the molecular mechanism of cellular reprogramming and opens the way to tackle the quest for the (up to date) evasive human TSC. http://dx.doi.org/10.1016/j.jri.2016.04.118 OP1 Uterine natural killer cell partnerships in early mouse decidua basalis A.M. Felker ∗ , B.A. Croy Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Canada Mouse decidua basalis (DB) is highly enriched in CD45+ leukocytes. In intact C57BL/6 (B6) DB examined at gestation day (gd)8.5 by whole mount in situ immunohistochemistry (WM-IHC), leukocyte conjugates are reported but nothing is known regarding their time-course, frequency, composition or importance. Virgin or gd3.5-9.5 B6 mice were studied by WM-IHC using anti-CD45, anti-Ly49C/I or DBA Lectin and one additional directly conjugated fluorescent antibody. Conjugates were analyzed by epifluorescence or confocal microscopy (n = 3 mice/time point). Confocal microscopy confirmed CD54+/CD11a+ immune synapses in CD45+ decidual cell conjugates. Conjugates are virtually absent prior to implantation, infrequent at gd5.5, but involve 19% of all CD45+ cells by gd8.5, then decline. Conjugation partners were defined for two uterine natural killer (uNK) cell subsets. Ly49C/I+DBA− uNK cells are the major subset before mating to gd6.5. At gd5.5/6.5, uNK cell conjugates involving Ly49C/I+ cells are abundant. By gd8.5/9.5, Ly49C/I−/DBA+ uNK cells are dominant as both homologous and heterologous conjugates. Both Ly49C/I+ and DBA+ cells strongly engage antigen presenting cells (CD11c+, CD68+ or MHCII+) at
Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Abstracts of invited and selected oral presentations
Opening lecture S1 Gene editing – From modeling diseases to treating patients T. Cathomen Medical Center – University of Freiburg, Germany Targeted genome editing with designer nucleases has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional “gene addition” type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise “gene surgery”. Moreover, designer nuclease technology allows for novel genetic interventions to fight infectious diseases, including chronic viral infections. We have developed highly specific designer nucleases based on transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. In preclinical models we demonstrate that TALENs and CRISPR-Cas9 nucleases can be employed to generate cells resistant to infection with human immunodeficiency virus type 1 (HIV-1) or to correct primary immunodeficiencies. Strategies to translate these findings into the clinic will be discussed. http://dx.doi.org/10.1016/j.jri.2016.04.117
Session 1: Models to understand the pathogenesis of pregnancy pathologies: from stem cells to animals S2 Trophoblast stem cells C. Kubaczka, H. Schorle ∗ Department of Developmental Pathology, Bonn University Medical School, Bonn, Germany Trophoblast stem cells (TSCs) are derived from polar trophectoderm (TE) or ectoplacental cone and represent the stem cell population of the extraembryonic lineage. TSCs have the ability to self-renew and give rise to all derivatives of the trophectoderm lineage. In recent years, we have begun to understand the molecular network governing TE and TSC-fate. In particular, the factors Tead4, Eomes, Cdx2, Tfap2c, Gata3 and Elf5 are thought to be crucial for 0165-0378/
TE/TSC-fate. Expressed in TE/TSCs, these genes cooperate to repress the somatic pluripotency program leading to specification, maintenance and fixation of the extraembryonic lineage in the early embryo. In an attempt to standardize the TSC-culture, we succeeded to develop a chemically defined media demonstrating, that the cytokines Fgf4 (plus Heparin) and TGF- are sufficient to keep TSC cultures in an undifferentiated state when grown on Matrigel coated dishes. Further, we were able to demonstrate, that overexpression of four factors (Tfap2c, Eomes, Gata3 and Ets2) suffices to reprogram murine fibroblasts to bona fide induced-TSC (iTSC). The iTSC were fully functional with almost identical expression profile and epigenetic signature when compared to TSC. These data provide the scaffold for a more detailed understanding of the molecular mechanism of cellular reprogramming and opens the way to tackle the quest for the (up to date) evasive human TSC. http://dx.doi.org/10.1016/j.jri.2016.04.118 OP1 Uterine natural killer cell partnerships in early mouse decidua basalis A.M. Felker ∗ , B.A. Croy Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Canada Mouse decidua basalis (DB) is highly enriched in CD45+ leukocytes. In intact C57BL/6 (B6) DB examined at gestation day (gd)8.5 by whole mount in situ immunohistochemistry (WM-IHC), leukocyte conjugates are reported but nothing is known regarding their time-course, frequency, composition or importance. Virgin or gd3.5-9.5 B6 mice were studied by WM-IHC using anti-CD45, anti-Ly49C/I or DBA Lectin and one additional directly conjugated fluorescent antibody. Conjugates were analyzed by epifluorescence or confocal microscopy (n = 3 mice/time point). Confocal microscopy confirmed CD54+/CD11a+ immune synapses in CD45+ decidual cell conjugates. Conjugates are virtually absent prior to implantation, infrequent at gd5.5, but involve 19% of all CD45+ cells by gd8.5, then decline. Conjugation partners were defined for two uterine natural killer (uNK) cell subsets. Ly49C/I+DBA− uNK cells are the major subset before mating to gd6.5. At gd5.5/6.5, uNK cell conjugates involving Ly49C/I+ cells are abundant. By gd8.5/9.5, Ly49C/I−/DBA+ uNK cells are dominant as both homologous and heterologous conjugates. Both Ly49C/I+ and DBA+ cells strongly engage antigen presenting cells (CD11c+, CD68+ or MHCII+) at