The effects of embryonic bodies on differentiation of mouse embryonic stem cells to neuronal cells in vitro / 生物医学工程学杂志

This study was directed at assessing the effects of embryonic bodies on differentiation of the embryonic stem cells. The embryonic bodies of different days gained by means of hanging, suspending and hung-suspended were processed by all-trans retinotic acids for 4 days. Then they were detected by immunocytochemistry methods and were measured to detect the fluorescence intensity changes of calcium after Glu stimulation. During this period, we observed the differentiation process and compared the differentiation ratio of embryonic stem cells processed by means of hanging, suspending for several days. It was found that the differentiation ratio of EBs obtained by suspending for 3 days, or by hanging for 3 days and suspending for 1 day, is relatively higher, which can help us find out the intrinsic differentiation mechanism of embryonic stem cells.

Isolation and differentiation of mouse embryonic stem cells

Recently, embryonic stem [ES] cells have become very important resources in basic medical researches. These cells can differetiate into derivatives of all primary germ layers. In order to isolate embryonic stem cells in vitro, the blastocyst were cultured and the morphological aspects, population doubling time, alkalin phosphatse and differentiation properties of the cells were investigated. The balstocysts from NMRI mice were cultured for 3 days up to time that inner cell mass [ICM] reach to the outgrowth stage. The cells were disaggregated and trypsinized every 3 days until the appearance of the colonies of ES cells. The colony positive cells were fixed and stained for alkaline phosphatase. The ES cells were cultured in suspension state for 5 days, at the same time Leukaemia Inhibitory Factor [LIF] was removed from media to form embryoid bodies[EBs]. The EBs were cultured for 8 - 20 days on collagen coated dish to induce the spontaneouse differentiation. During the 6-9 days after the disaggregation of ICM in the expansion stage, the colony of ES cells appeared as a flat monolayer mass with strike boundaries and nondistinguish cytoplasm including a few nuclei. In colony formation stage, the morphology changed from flat monolayer to round multilayer with strike define boundaries. Undifferentiated cells were seen as intensely small cells attached together compactly with high nucleus/cytoplasm [N/C] ratio. The cells of colonies tend to differetiate by separation from each other and became larger and diffused on substrate by attaching to dish. The positive alkaline phosphatase cells were seen in typical morphology of ES colonies. The EBs cells were seen in culture after 5 days in suspension and began to spontaneously differentiate into various types of cells such as nerve and hematopoitic lineages.Despite strike morphology of ES colonies, it is difficult to distinguish the differentiated from undifferentiated cell colonies in the colony formation stage. New ES cells are capable to give rise into EBs and are susceptible of spontaneously differentiation in various type of cells

Efficient Induction of Dopaminergic Neurons from Embryonic Stem Cells for Application to Parkinson's Disease

For cell replacement therapy of neurodegenerative diseases such as Parkinson's disease (PD), methods for efficiently generating midbrain dopaminergic (DA) neurons from embryonic stem (ES) cells have been investigated. Two aspects of DA neuron generation are considered: genetic modification and manipulation of culture conditions. A transcription factor known as critical for development of DA neurons, Nurr1, was introduced into ES cells to see how they facilitate the generation of DA neurons from ES cells. Also, two culture procedures, the 5-stage method and stromal cell-derived inducing activity (SDIA) method, were used for ES cell differentiation. Using the 5-stage method, we and others previously demonstrated that Nurr1-overexpressing ES cells, under treatment of signaling molecules such as SHH and FGF8 followed by treatment of ascorbic acid, can differentiate into DA neurons with a high efficiency (> 60% of TH+/Tuj1+ neurons). Furthermore, using the SDIA method with treatment of signaling molecules, we found that Nurr1-overexpressing ES cells can differentiate to DA neurons with the highest efficiency ever reported (~90% of TH+/Tuj1+ neurons). Importantly, our semi-quantitative and real-time PCR analyses demonstrate that all known DA marker genes (e.g., TH, AADC and DAT) were up-regulated in Nurr1- overexpressing ES cells when compared to the na ve ES cells. These cells produced increased dopamine compared to na ve D3 cells after differentiation. In the in vivo context after transplantation, the genetically modified ES cells also showed the highly increased dopaminergic neuronal phenotypes. Thus, the combination of genetic engineering and appropriate culture conditions provides a useful tool to generate a good cell source from ES cells for cell replacement therapy of degenerative diseases such as PD.

Agrobacterium rhizogenes vs auxinic induction for in vitro rhizogenesis of Prosopis chilensis and Nothofagus alpina

The induction and improvement of in vitro rhizogenesis of microshoots of Prosopis chilensis (Mol.) Stuntz and Nothofagus alpina (Poep. et Endl. Oerst.) were compared using Agrobacterium rhizogenes (Ar) versus indole-3-butyric acid (IBA) in the culture media. Microshoots of P. chilensis (1-2 cm length), coming from in vitro grown seedlings, were cultivated in a modified Broadleaved Tree Medium (BTMm) containing half salt concentration of macronutrients and 0.05 mg x L(-1) benzilaminopurine (BAP). After 30 days, microshoots with 2-4 leaves were selected and cultured in BTMm-agar in presence or abscense of Ar and in combination with IBA. For N. alpina, the apical shoots with the first 2 true leaves, from 5 weeks old seedlings, were cultured in the abovementioned medium, but with 0.15 mg x L(-1) of BAP. After 2 months, microshoots with 2-3 leaves were selected and cultured in BTMm-agar, supplemented with 5 mg x L(-1) IBA or in liquid BTMm on perlite and, in the presence or absence of A. rhizogenes (Ar) and in combination with 3 mg x L(-1) IBA. Rooting in P. chilensis reached 100.0% when Ar infection was produced in the presence of IBA, increasing both, the number and dry weight of roots. In N. alpina, 90.0% of rooting efficiency was obtained when Ar infection was produced in liquid culture and in the absence of auxin.

Influence of the neural tube/notochord complex on MyoD expression and cellular proliferation in chicken embryos

Important advances have been made in understanding the genetic processes that control skeletal muscle formation. Studies conducted on quails detected a delay in the myogenic program of animals selected for high growth rates. These studies have led to the hypothesis that a delay in myogenesis would allow somitic cells to proliferate longer and consequently increase the number of embryonic myoblasts. To test this hypothesis, recently segmented somites and part of the unsegmented paraxial mesoderm were separated from the neural tube/notochord complex in HH12 chicken embryos. In situ hybridization and competitive RT-PCR revealed that MyoD transcripts, which are responsible for myoblast determination, were absent in somites separated from neural tube/notochord (1.06 and 0.06 10-3 attomol MyoD/1 attomol á-actin for control and separated somites, respectively; P<0.01). However, reapproximation of these structures allowed MyoD to be expressed in somites. Cellular proliferation was analyzed by immunohistochemical detection of incorporated BrdU, a thymidine analogue. A smaller but not significant (P = 0.27) number of proliferating cells was observed in somites that had been separated from neural tube/notochord (27 and 18 for control and separated somites, respectively). These results confirm the influence of the axial structures on MyoD activation but do not support the hypothesis that in the absence of MyoD transcripts the cellular proliferation would be maintained for a longer period of time

Transferencia embrionaria en día 3 versus día 5 bajo sistema de cultivo secuencial / Day 3 versus Day 5 Embryonic transference under sequential culture system

El presente tiene como objeto validar la hipótesis que el cultivo embrionario en día 5 y la transferencia en blastocisto es superior a la convencional en día 3. Se analizan los resultados obtnidos sobre un total de 39 ciclios con recuperación ovacitaria en los que se realizaron 36 transferencias, 22 en día 3 y 14 en día 5 en población no seleccionada y bajo las mismas condicions de cultivo. Ambsa muestras no presentan diferencias en su perfil poblaconal y parámetros de respusta a la estimulación

La transferencia de embriones. Una técnica de mejoramiento animal en ganado de lidia / Embryo transfer. A technique of animal improvement in fighting livestock

Dos explotaciones del estado de Tlaxcala (uno productor de ganado de lidia y otro productor de leche), se utilizaron para evaluar dos tipos de hormonas, la gonadotropina de suero de yegua gestante (PMSG) y la hormona folículo estimulante (FSH), en relación con la cantidad y calidad de embriones que se obtuvieron por superovulación en el programa de transferencia de embriones. Para la superovulación, se formaron dos grupos de cinco hembras de lidia; administrando al primero, FSH en dosis descendentes de 100 U.I. a 37.5 U.I. durante cuatro días consecutivos, y al segundo grupo con la administración de PMSG, en una sola dosis de 2,000 U.I. Se obtuvieron con FSH, 25 embriones transferibles, 20 degenerados o inmaduros y 15 ovocitos no fertilizados, sin embargo con PMSG, se obtuvieron cinco embriones transferibles, cinco degenerados o inmaduros y cinco ovocitos no fertilizados, lo que indica que el tratamiento con FSH, es más efectivo que el de PMSG para superovular a hembras de lidia con una probabilidad estadísticamente significativa (P < 0.05).Los 30 embriones que se obtuvieron para ser transferidos, fueron transportados a la explotación de leche, donde se llevó a cabo la transferencia de los mismos a 30 vaquillas respectivamente, mismas que fueron consideradas como receptoras. Del total de las transferencias, sólo cinco vaquillas se diagnosticaron como gestantes (16.6 por ciento), coincidiendo con embriones obtenidos de hembras superovuladas con FSH; cinco (16.6 por ciento) repitieron el estro a los 21 días de ciclo; otras cinco vaquillas (16.6 por ciento) repitieron el estro hasta los 37 días de ciclo; diez (33.3 por ciento) sólo presentaron un cuerpo lúteo en el ovario derecho y las cinco restantes (16.6 por ciento) además de presentar también un cuerpo lúteo en el ovario derecho, presentaban infección uterina. Esto se corroboró estadísticamente con una probabilidad estadística (P < 0.05).

relationship between plasma progesterone concentration and embryo production in dairy cows superovulated with FSH or PMSG

Fourteen primiparous lactating cows, 3.5 to 4.5 years old in a military dairy farm nearby Cairo [Egypt] were used in this study. Following synchronization, the animals were randomly assigned into two groups; the first group [6 cows] was superovulated with a total dose of 32 mg FSH-P whereas the second group [8 cows] was treated with 3000 iu PMSG. Embryos were collected non-surgically on day 7 [day of estrus = day 0]. Plasma samples were taken from cows of both groups on day-4 [first day of gonadotrophin treatment], day-2 [day of PGF[2] alpha injection], day 0 [day of estrus] days 1,3,5 post-estrus and day 7 [day of recovery] for progesterone assay. The results revealed that FSH was more effective as a superovulatory agent than PMSG. Higher [P < 0.05] ovulalion rate [11.33 +/- 1.85 vs 6.63 +/- 1.14], number of embryos recovered per donor [5.17 +/- 1.20 vs 2.44 +/- 0.45] as well as number of transferable embryos [4.16 +/- 1.01 vs 1.86 +/- 0.42] were obtained from FSH than from PMSG-treated cows. However, more follicles [> 10 mm] were palpated in the PMSG-treated group [2.00 +/- 0.40 vs 0.33 +/- 0.19; P < 0.01]. Coefficients of correlation between progesterone concentration at initiation of superovulalion and each of the ovulation rate, number of embryos recovered and number of transferable embryos were higher in FSH than in PMSG-treated cows. When the data of both groups were pooled, the respective correlations [0.74, 0.69, 0.67] were still highly [P < 0.01] significant. Moreover, significant correlations were estimated between progesterone concentration and the mentioned parameters in FSH group at the day of recovery. This study revealed that measurement of plasma progesterone concentration can serve as a prognostic tool to predict the yield of fertile eggs and quality of embryos

Los inositoles de la membrana celular y el efecto del Li+ sobre el desarrollo embrionario / The inositols of the cellular membrane and the effect of Li+ in the development of the embryo

El Li+ tiene la capacidad, en embriones sanos, de inducir la formación supernumeraria de estructuras cefálicas y de revertir las deficiencias del eje neural que se evidencian en embriones expuestos a la luz ultravioleta. Por otra parte, la hidrólisis de los fosfoinositodos de la membrana se ha implicado en los aumentos de Ca2 que proceden a los eventos que se suceden durante la fertilización y el desarrollo embrionario y el Li+ impide que los niveles de fosfoinositol (4,5), bifosfato retornen a sus niveles normales. La conclusión a la cual se debe llegar es que el Li+ altera la reespecificación dorsoanterior de los embriones y que esto debe depender de su capacidad de interferir con el ciclo de los fosfoinositidos. Estas observaciones tienen relevancia particular puesto que los efectos específicos del Li+ sobre el ciclo de los fosfoinositidos permiten suponer que alguno de los intermediarios de este ciclo debe jugar un papel importante en la diferenciación y especificación dorsoanterior del embrión. Es preocupante el hecho de que estos efectos pueden obtenerse utilizando concentraciones de Li+ similares a las usadas terapéuticamente en el manejo de las enfermedades maniacodepresivas

Aspectos médico-legales de la fecundación in vitro y transferencia embrionaria FIVET: primera parte / Medicolegal aspects on In-vitro fertilization and embryonic transference FIVET: first part

La fecundación in vitro o fecundación extrauterina con transferencia embrionaria (FIVET) fue una realidad en julio de 1978 cuando Edwards y Steptoe lograron en Inglaterra el nacimiento de una niña concebida fuera del claustro materno. Desde entonces no se han aclarado problemas legales como los derechos del embrión, el deber del Estado de protegerlo y el interés público en la prevención de niños genéticamente defectuosos. Se considera que solamente deben fecundarse aquellos óvulos que serán implantados, a fin de evitar el problema de embriones sobrantes. El procedimiento debe estar bajo control de un comité consultor de médicos, biólogos, moralistas, abogados, genetistas y autoridades del Ministerio de Salud. El médico practicante de la FIVET tiene responsabilidad penal y civil por su deber de diligencia en el examen de los componentes germinales y de los progenitores, así como en la custodia del embrión. El lavado del útero para remover un embrión que se trate de implantar en otra mujer debe constituir delito de aborto. Las donaciones de gametos deben limtar se por ley para prevenir el riesgo de relaciones incestuosas. La dignidad del ser humano como valor fundamental de la sociedad es enfatizado. PALABRAS CLAVES: Fecundación in vitro, transferencia embrionaria, genética y ley, medicina legal, derechos del embrión.

Mechanisms underlying the organizer formation in Bufo arenarum embryos

El mesodermo prospectivo en la gástula temprana de Bufo arenarum fue previamente identificado ocmo una banda marginal de células grises. Para analizar su capacidad de diferenciación, explantos de estas células, fueron cultivados en el interior de vesículas ectodermales, en aislamiento y en combinación con componentes vegetativos. Cuando se cultivaron en aislamiento, fragmentos dorsales y ventrales de la zona marginal profunda se comportaron diferentemente. Mientras que los explantos ventrales produjeron células sanguíneas, los explantos dorsales fallaron en diferenciar permenecidendo como masas de células ricas en vitelo. Por otra parte, ambos cultivos fueron drásticamente modificados cuando se asociaron con células superficiales de la zona blastoporal, las que causaron los siguiente s efectos. a) Promoción de diferenciación en exploantos marginales dorsales, capaces ahora de generar estructuras notocordales y somáticas en adición a células mesenquimáticas. b) Promoción de dorsalización en explantos marginales ventrales, los cuales cambiaron su destino esperando desarrollando componentes axiales, similares a aquellos producidos por explantos dorso marginales "activados". Por el contrario, cultivos combinados de piezas animales y vegetativas fueron incapaces de generar estructuras mesodermales. Estos estudios sugieren que el mesodermo axial, identificado como el "organizador", se desarrolla a partir de un sustrato marginal de células mesodérmicas genuinas, mediante un estímulo inductivo dorsalizante originado en células periblastoporales superficiais

Analysis of the mechanisms involved in neural induction

La determinación del área neural presuntiva en el embrión de vertebrado tiene lugar durante la gastrullación bajo la influencia inductora del cordomesoblasto en el curso de su invaginación. A pesar de los extensivos estudios destinados a esclarecer este complejo proceso, el problema no ha llegado a ser resuelto. En el presente artículo se consiera principalmente la competencia de la célula efectora y la naturaleza de la señal mediadora en este proceso de interación celular. Ambos puntos son discutidos en base a datos experimentales obtenidos principalmente de embriones de anfibios