RESUMEN
Human leukocyte Ag-G, a tolerogenic molecule that acts on cells of both innate and adaptive immunity, plays an important role in tumor progression, transplantation, placentation, as well as the protection of the allogeneic fetus from the maternal immune system. We investigated HLA-G mRNA and protein expression in human embryonic stem cells (hESC) derived from the inner cell mass (ICM) of blastocysts. hESC self-renew indefinitely in culture while maintaining pluripotency, providing an unlimited source of cells for therapy. HLA-G mRNA was present in early and late passage hESC, as assessed by real time RT-PCR. Protein expression was demonstrated by flow cytometry, immunocytochemistry, and ELISA on an hESC extract. Binding of HLA-G with its ILT2 receptor demonstrated the functional active status. To verify this finding in a physiologically relevant setting, HLA-G protein expression was investigated during preimplantation development. We demonstrated HLA-G protein expression in oocytes, cleavage stage embryos, and blastocysts, where we find it in trophectoderms but also in ICM cells. During blastocyst development, a downregulation of HLA-G in the ICM cells was present. This data might be important for cell therapy and transplantation because undifferentiated hESC can contaminate the transplant of differentiated stem cells and develop into malignant cancer cells.
Asunto(s)
Masa Celular Interna del Blastocisto/inmunología , Masa Celular Interna del Blastocisto/metabolismo , Células Madre Embrionarias/inmunología , Células Madre Embrionarias/metabolismo , Antígenos HLA/biosíntesis , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos CD/metabolismo , Masa Celular Interna del Blastocisto/citología , Línea Celular Tumoral , Células Cultivadas , Fase de Segmentación del Huevo/citología , Fase de Segmentación del Huevo/inmunología , Fase de Segmentación del Huevo/metabolismo , Regulación de la Expresión Génica/inmunología , Antígenos HLA/genética , Antígenos HLA/metabolismo , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Tolerancia Inmunológica/genética , Receptor Leucocitario Tipo Inmunoglobulina B1 , Oocitos/inmunología , Oocitos/metabolismo , Unión Proteica/genética , Unión Proteica/inmunología , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Inmunológicos/metabolismoRESUMEN
BACKGROUND: Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS: Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS: Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT-PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION: We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent.
Asunto(s)
Blastómeros/citología , Técnicas de Cultivo de Embriones , Células Madre Embrionarias , Línea Celular , Desarrollo Embrionario , Humanos , Células Madre PluripotentesRESUMEN
Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great promise for tissue transplantation. One of the rising techniques that allows studying the differentiation state of ES cells is quantitative RT-PCR (qRT-PCR). When relative quantification by qRT-PCR is applied, accurate normalization is necessary, since differentiated embryonic stem cells and developing embryos contain heterogeneous cell populations. Corrections for variations in the qRT-PCR reaction are needed to allow comparisons between different samples. We applied the normalization tools geNorm and Normfinder to ten reference genes identifying the most stable ones for relative quantification of gene expression during differentiation of human ES cells, as well as in differentiated mouse ES cells and in the developing mouse embryo. For relative quantification by qRT-PCR in these systems, we advise to use normalization factors based on multiple stable reference genes. However, when the use of several reference genes would be unpractical, a single reference gene in each experimental setup could be sufficient. When looking for single stable reference genes, beta-actin works best in both mouse embryo and ES cell experiments and glyceraldehyde-3-phosphate-dehydrogenase can be applied in both mouse and human ES cell experiments.