Defining the genomic signature of totipotency and pluripotency during early human development.

The genetic mechanisms governing human pre-implantation embryo development and the in vitro counterparts, human embryonic stem cells (hESCs), still remain incomplete. Previous global genome studies demonstrated that totipotent blastomeres from day-3 human embryos and pluripotent inner cell masses (ICMs) from blastocysts, display unique and differing transcriptomes. Nevertheless, comparative gene expression analysis has revealed that no significant differences exist between hESCs derived from blastomeres versus those obtained from ICMs, suggesting that pluripotent hESCs involve a new developmental progression. To understand early human stages evolution, we developed an undifferentiation network signature (UNS) and applied it to a differential gene expression profile between single blastomeres from day-3 embryos, ICMs and hESCs. This allowed us to establish a unique signature composed of highly interconnected genes characteristic of totipotency (61 genes), in vivo pluripotency (20 genes), and in vitro pluripotency (107 genes), and which are also proprietary according to functional analysis. This systems biology approach has led to an improved understanding of the molecular and signaling processes governing human pre-implantation embryo development, as well as enabling us to comprehend how hESCs might adapt to in vitro culture conditions.

Comparison of Cryotip vs. Cryotop for mouse and human blastomere vitrification.

OBJECTIVE: Compare the efficiency of two vitrification methods for single-blastomere cryopreservation with mouse or human embryos. DESIGN: Experimental prospective controlled study. SETTING: Research center. PATIENT(S): Human Blastomeres were obtained after biopsy. INTERVENTION(S): Mouse blastomeres were obtained after diluting the zona pellucida of embryos with Tyrode acid and manual isolation. Individual human blastomeres were biopsied from embryos following established clinical protocols. The modified open Cryotop and classical closed Cryotip vitrification systems were assayed. After thawing, some mouse blastomeres were fixed and analyzed for apoptotic markers annexin V and caspase 3 with the use of immunofluorescence and confocal microscopy. Ultrastructural morphology was examined using electron microscopy. The human blastomere division rate was assessed 24 hours after thawing. MAIN OUTCOME MEASURE(S): Blastomere survival and division rates after thawing, apoptotic markers, and electron microscopy; adhesion and outgrowth rates of human blastomeres. RESULT(S): After thawing, survival rates in mouse blastomeres using Cryotop vs. Cryotip were 38.46% vs. 85.41%, respectively. As expected, thawed morphologically alive blastomeres were classified negative for annexin V and caspase 3, whereas degenerated blastomeres were positive for both. Further, nuclear chromatin was compacted. Survival rates of human blastomeres vitrified with Cryotop were 22.78% vs. 53.77% with Cryotip. Division capabilities were 16.6% and 47.16%, respectively, in Cryotop and Cryotip. CONCLUSION(S): The closed system is more efficient in preserving mouse and human blastomeres in terms of acceptable survival and division rates, and it also complies with European Union directives.

Derivation, characterization, differentiation, and registration of seven human embryonic stem cell lines (VAL-3, -4, -5, -6M, -7, -8, and -9) on human feeder.

Derivation of human embryonic stem cell lines has been a remarkable scientific achievement during the last decade. Human embryonic stem cells are regarded as an unlimited cell source for replacement therapy in regenerative medicine. Clearly, the scientific community requires proper derivation, characterization, and registration with the purpose of making them available for research and future medical applications worldwide. In this paper, we report our derivation work as the Valencian Node of the Spanish Stem Cell Bank in the generation, characterization, and registration of VAL-3, -4, -5, -6M, -7, -8, and 9 (www.isciii/htdocs/terapia/terapia_bancocelular.jsp). The derivation process was performed on microbiologically tested and irradiated human foreskin fibroblasts and designed to minimize contact with xeno-components in knockout Dulbecco's modified Eagle's medium supplemented with knockout serum replacement and basic fibroblast growth factor. Fingerprinting of the cell lines was performed to allow their identification and traceability. All lines were expressed at the mRNA and specific protein markers for undifferentiation and were found to be negative for classical differentiation markers such as neurofilament heavy chain (ectoderm), renin (mesoderm), and amylase (endoderm). All lines displayed high levels of telomerase activity and were shown to successfully overcome cryopreservation and thawing. Finally, we demonstrated the potential to differentiate in vitro (embryoid body formation) and in vivo (teratoma formation) into cell types from all three germ layers. Teratoma derived from all human embryonic stem cell lines present similar morphological features except VAL-8 that display more aggressive tumor behavior with a larger proportion of solid tissues, as opposed to cyst formation in the other cell lines.

Insights on blastomere nuclearity.

PURPOSE: To analyze the results of our transferred embryos, especially those that "changed" their blastomere nuclearity from Multinucleated (MN) to Mono-nucleated during development. METHODS: Pregnancies where at least one MN embryo was transferred were retrospectively evaluated and categorized in order to record and follow-up on the ones that were implanted. Embryos were classified as normal (when all blastomeres were mono-nucleated on day one and two of development), corrected (multinucleated embryos on day one that became mono-nucleated on day two) and non-corrected (multinucleated either on day one, on day two or both days). RESULTS: There were 633 transfer cycles analyzed. Thirty-three percent (206) had at least one embryo with a MN blastomere at a given stage of development. Pregnancy and implantation rates were 29.0% and 19.0% for the group of exclusively mono-nucleated embryo transfers, and 28.6% and 15.8% for the group with at least one MN embryo transferred. The pregnancy outcome for "corrected" and "non-corrected" embryos could be corroborated unequivocally in only 9 cases, with an outcome of 8 and 4 normal babies, respectively. CONCLUSIONS: Because the amount of data analyzed is not satisfactorily large, differences were not significantly different; however, a trend may exist showing that normal at term pregnancies obtained from corrected embryos are more likely to occur than those from non-corrected embryos. Nuclear observation on a daily basis should be one of the strategies used to select the best embryos for transferring, to improve implantation rates and avoid multiple pregnancies.

Monochorionic triplets after single embryo transfer.

A 40-year-old patient underwent intracytoplasmic sperm injection and assisted hatching, and a single embryo was transferred. Ultrasonography demonstrated a single gestational sac containing monochorionic tri-amniotic pregnancy. Several factors that have been implicated in the aetiology of monozygotic triple pregnancies after IVF appear to be present in this case. To avoid multiple pregnancies after IVF, it is time to have definite predictive factors for the occurrence of monozygotic multiple pregnancies as well as transferring only a single embryo.

Efecto de la concentración dietaria de la levadura (Saccharomyces carlsbergensis) recuperada de la cerveza, en pollos macho Warren / Effect of dietary concentration of Saccharomyces carlsbergensis yeast recovered from beer, in Warren male chicks

Con el objetivo de establecer el nivel máximo de sustitución del aislado proteínico de soya por la proteína de la levadura Saccharomyces carlsbergensis, recuperada de la cerveza, con los menores efectos metabólicos relacionados, se alimentarón por 15 días un total de 42 pollos macho (Warren), de 1 día de edad, divididos en seis grupos con siete animales cada uno. Cada grupo recibió una de las dietas con 0%, 25%, 50%, 75% y 100% del suplemento proteínico basado en proteína de levadura, adicionada a expensas del aislado proteínico de soya. A fin de estimar el valor NPR de la proteína de la levadura, se incluyó también un grupo, el cual recibió una dieta libre de proteínas. Se determinaron la utilización proteínica y los cambios de lípidos totales, triglicéridos y colesterol, tanto en el plasma como en el hígado. En los grupos alimentados con 75% y 100% de proteína de levadura, se observó un descenso en el crecimiento y un incremento en las concentraciones hepáticas y renales del ácido úrico, si bien el consumo de la dieta no se modificó sustancialmente. Así, la utilización de proteína medida como PER y NPR, fue menor en estos grupos. El ácido úrico plasmático no se modificó en ninguno de los grupos. Los lípidos plasmáticos tampoco se modificaron a ninguna concentración de levadura, mientras que en el hígado, los lípidos totales disminuyeron, así como los triglicéridos, al incrementarse la levadura dietaria. Los resultados indicaron que le nivel máximo de sustitución de la proteína de levadura usando células completas en dietas iniciadoras para pollos, es del 50%