title: Liver sinusoidal endothelial cells support the survival and undifferentiated growth of the CGR8 mouse embryonic stem cell line: possible role of leukemia inhibitory factor (LIF).
abstract: Murine embryonic stem cells (muESC) are maintained and expanded in vitro by culturing in the presence of leukemia inhibitory factor (LIF) or by coculturing on murine embryonic fibroblast (MEF). Previously we have shown that liver sinusoidal endothelial cells (LSEC) promote the survival, proliferation and differentiation of hematopoietic stem cells. In the present study we investigated whether LSEC might promote the survival and undifferentiated growth of muESC. For these purposes, muESC (CGR8 cell line) were cultured on LSEC monolayers (muESC/LSEC) or in the presence of conditioned medium from LSEC cultures (muESC/LSEC-CM), both in the absence of LIF. Microscopic observation showed the growth of undifferentiated ESC colonies in both muESC/LSEC or muESC/LSEC-CM cultures. A significant reduction in the growth of undifferentiated ESC colonies was observed when ESC were cultured in LSEC-CM previously incubated with anti-LIF. RT-PCR and Western blot analysis showed that LSEC constitutively express LIF at the mRNA and protein level. At different times of culture, muESC were harvested and analyzed for the expression of embryonic markers (SSEA-1 and Oct-4) and differentiation capacity. Flow cytometry analysis showed the presence of a higher percentage of muESC (>90%) expressing SSEA-1 in muESC/LSEC-CM, as compared with muESC/LSEC cocultures. muESC obtained from both types of cultures formed embryoid bodies in vitro, and form teratomas in testicles of mice. These results provide the first evidence that LSEC support the in vitro survival, self-renewal, undifferentiated growth and differentiation capacity of the muESC CGR8 cell line.
authors.affiliation: Unidad de Terapia Celular–Laboratorio de Patología Celular y Molecular, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Silva-Cote
authors.forename: Jose E
authors.initials: JE
authors.lastname: Cardier
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned / Q000494:pharmacology; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D053605:Leukemia Inhibitory Factor / Q000235:genetics / Q000378:metabolism*; D008099:Liver / Q000166:cytology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013724:Teratoma / Q000378:metabolism / Q000473:pathology; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D019138:Antibodies, Blocking; D015415:Biomarkers; D017077:Culture Media, Conditioned; D053605:Leukemia Inhibitory Factor; D012333:RNA, Messenger; D014157:Transcription Factors