Organoides: fundamentos, presente y futuro / Organoids: fundamentals, present and future

RESUMEN Los organoides son estructuras miniaturizadas, generadas principalmente a partir de células madre pluripotentes inducidas, que se cultivan en el laboratorio conservando sus características innatas o adquiridas. Tienen el potencial de reproducir procesos de desarrollo biológico, modelar procesos patológicos que permitirán el descubrimiento de nuevos fármacos y propicien la medicina regenerativa. Sin embargo, estas experiencias requieren perfeccionamiento constante porque pueden haberse realizado variaciones en la constitución de estos órganos. Por ello, el presente artículo tiene como objetivo revisar la información actualizada sobre organoides y sus procesos experimentales básicos y recientes, empezando por la gastrulación, para tratar de imitar, en lo posible, la formación de las tres capas: ectodermo, mesodermo y endodermo, incluyendo los factores que intervienen en la inducción, diferenciación y maduración en la generación de estos organoides. Asimismo, el diseño y preparación de medios de cultivo altamente especializados que permitan obtener el órgano seleccionado con la mayor precisión y seguridad. Se realizó una búsqueda de artículos originales y de revisión publicados en PubMed, Nature y Science. Los artículos se seleccionaron por sus resúmenes y por su texto completo. Las conclusiones de este articulo destacan las ventajas futuras en el uso y aplicaciones de los organoides.

ABSTRACT Organoids are tiny structures, mainly generated from induced pluripotent stem cells, which are cultured in the laboratory while retaining their innate or acquired characteristics. They have the potential to reproduce biological development processes, model pathological processes that will enable the discovery of new drugs and promote regenerative medicine. However, these processes require constant improvement because variations may have occurred in the constitution of the organs. Therefore, this article aims to review updated information on organoids and their basic and recent experimental processes, starting with gastrulation, in an attempt to mimic, as much as possible, the formation of the three layers: ectoderm, mesoderm and endoderm; as well as the information regarding the factors involved in the induction, differentiation and maturation during the generation of organoids. Likewise, the design and preparation of highly specialized culture media that allow obtaining the selected organ with the highest precision and safety. We searched for original and review articles published in PubMed, Nature and Science. Articles were selected for their abstracts and full text. The conclusions of this article highlight the future advantages in the use and applications of organoids.

Understanding of Human Embryo Development for Teratogen Counselling

Human embryology is the study of development from a single cell to a baby in 9 months. Implantation occurs at the end of the first week of development. The second week of development is known as the week of 2's. Gastrulation, the most characteristic event occurring in the third week, establishes three germ layers composed of ectoderm, mesoderm, and endoderm. The three germ layers and neural crest cells lead to the development of their own tissues and organs during the embryonic period, which extends from the third to the eighth week. Major congenital malformations occur in the embryonic period. The fetal period, from the third month to the day of birth, is the time for maturation of tissues and organs, and growth of the body. Because of the close relationship between embryology and congenital abnormalities, knowledge of human development is essential to assess the effects on the embryo when the mother has been exposed to teratogens. This paper briefly reviews the normal embryonic development and associated congenital malformation.

Expression analysis of ciliary rootlet coiled coil protein mRNA during Xenopus development / 대한수의학회지

Ciliary rootlet coiled coil protein (CROCC), the structural component that originates from the basal body at the proximal end of the ciliary rootlet, plays a crucial role in maintaining the cellular integrity of ciliated cells. In the current study, we cloned Xenopus CROCC and performed the expression analysis. The amino acid sequence of Xenopus laevis was related to those of Drosophila, cow, goat, horse, chicken, mouse and human. Reverse transcription polymerase chain reaction analysis revealed that CROCC mRNA encoding a coiled coil protein was present maternally, as well as throughout early development. In situ hybridization indicated that CROCC mRNA occurred in the animal pole of embryo during gastrulation and subsequently in the presumptive neuroectoderm at the end of gastrulation. At tailbud stages, CROCC mRNA expression was localized in the anterior roof plate of the developing brain, pharyngeal epithelium connected to gills, esophagus, olfactory placode, intestine and nephrostomes of the pronephric kidney. Our study suggests that CROCC may be responsible for control of the development of various ciliated organs.

Na, K-ATPase beta2 isoform (atp1b2) expressed in the retina of Xenopus / 동물의과학연구지

The ubiquitous Na, K-ATPase is a membrane-bound ion pump located in the plasma membrane in all animal cells and plays an essential role in a variety of cellular functions. Studies in several organisms have shown that this protein regulates different aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Na, K-ATPase is an important factor for retinal development, and combinations of the isoforms of each of its subunits are expressed in different cell types and determine its functional properties. In this study, we performed RT-PCR assay to determine temporal expression and in situ hybridization to determine spatial expression of Na, K-ATPase beta2 isoform (atp1b2) in Xenopus laevis. Focusing on retinal expression to distinguish the specific expression domain, we used retinal marker genes sox4, sox11, vsx1, and . Xenopus atp1b2 was expressed from late gastrulation to the tadpole stage. Using whole mount in situ hybridization, we showed that Xenopus atp1b2 was expressed broadly in the eye, the whole surface ectoderm, and gills. In situ hybridization on sections revealed detailed and specific expression in the outer nuclear layer of the retina, which consists of two major classes of photoreceptors, rods and cones, surface ectoderm, pharyngeal epithelium, and gills. These findings indicate that atp1b2 may play an important role for the development of Xenopus retina.

Desarrollo embrionario del capaz pimelodus grosskopfii (Steindachner, 1879) / Embryonic development of capaz pimelodus grosskopfii (Steindachner, 1879)

El conocimiento del desarrollo embrionario en los peces es especialmente importante en especies nativas con potencial para la piscicultura, en virtud que permite identificar eventos morfológicos y cronológicos, necesarios para establecer prácticas de manejo durante las fases de incubación y larvicultura. El capaz (Pimelodus grosskopfii) es una especie con potencial para cultivo comercial, por sus hábitos alimenticios omnívoros y aceptación de su carne en el mercado. Para estudiar el desarrollo embrionario de la especie, ejemplares adultos sexualmente maduros fueron inducidos a la reproducción con extracto de hipófisis de carpa (5,75 y 4,0 mg Kg-1, hembras y machos, respectivamente). Los óvulos seminados fueron incubados en un sistema de flujo ascendente de 30 L a 27 +/- 1 °C. Las muestras (n=30) fueron colectadas al momento de la extrusión, durante la fertilización y cada 15 minutos a partir de las 0 horas postfertilización (HPF) hasta las 2 horas y cada 30 minutos desde las 2 HPF hasta 5 HPF; finalmente, entre las 5 HPF y la eclosión, cada 60 minutos. Los óvulos fertilizados presentaron forma esférica, sin adherencias y con amplio espacio perivitelino. El desarrollo embrionario finalizó a las 12 HPF. La diferenciación del polo animal y vegetal ocurrió a las 0,2 HPF, el primer clivaje a las 0,3 HPF, el blastodisco alto y estratificado a las 1,8 HPF, el blastodisco achatado a las 3,3 HPF, la epibolia < a 50 por ciento se observó a las 4 HPF, el cierre del blastoporo a las 5,7 HPF, la diferenciación cráneo caudal e inicio de la neurolación a las 7 HPF, la diferenciación de las vesículas ópticas, óticas y vesícula de Kupffer a las 8,5 HPF, la liberación de la cola del vitelo a las 10 HPF, los primeros movimientos se observaron a las 10,5 HPF y finalmente la eclosión ocurrió a las 12 HPF. Las larvas al eclosionar presentaron una longitud total de 2987+/-67 um, sin pigmentación, tracto digestivo rudimentario, sin abertura bucal ni anal y presencia de cromatóforos...

The knowledge of embryonic development in fish is important in native species with potential for fish farming, by virtue of which it makes possible to identify morphological and chronological events to establish management practices during incubation periods and larviculture. The capaz (Pimelodus grosskopfii) is a species with potential for commercial crop, due to their omnivorous eating habits and acceptance of its meat in the market. To study the embryonic development of the species, sexually mature adult specimens were induced to reproduce with carp pituitary extract (5.75 and 4.0 mgKg-1, females and males, respectively). The inseminated oocytes were incubated in an upward flow system 30 a 27 +/- 1 ° C. The samples (n = 30) were collected at the same time of the extrusion, during fertilization, and every 15 minutes starting from 0 to 2 hours post fertilization (HPF) and every 30 minutes from 0 to 2 HPF, and every 30 minutes from 2 to 5 HPF; finally, between 5 HPF and hatching every 60 minutes. The fertilized oocytes had a spherical shape without adhesions and large perivitelline space. Embryonic development took 12 HPF. The differentiation in animal and vegetal pole occurred at 0.2 HPF, the first cleavage at 0.3 HPF, stratified and high blastodisc at 1.8 HPF, flattened blastodisc at 3.3 HPF, the epiboly <50 percent was observed at 4 HPF, the closure of the blastopore at 5.7 HPF, cranial-caudal differentiation and starting the neurolation at 7 HPF, the differentiation of the optic vesicles, otic and Kupffer's vesicle at 8.5 HPF, tail of the vitelum was released at 10 HPF, first movements were observed at 10.5 and finally hatching occurred at 12 HPF. When the larvae hatched, they showed a total length of 2987+/-67 µm, without depigmentation, rudimentary digestive system without oral and anal opening and the presence of chromatophores on the yolk sac.

Desarrollo embrionario del sistema nervioso central y órganos de los sentidos: revisión / Embryonic central nervous system and sense organ development: review

El sistema nervioso tiene origen en la capa germinal ectodérmica. Así como en la vida posnatalel sistema nervioso está claramente diferenciado en sistema nervioso central y periférico,en su etapa embrionaria la formación de cada uno sigue caminos diferentes, pero cercanos.Esta revisión hace hincapié en la formación del sistema nervioso central y de estructurasespecíficas, como los órganos de los sentidos. Se presenta un breve recorrido por procesoscomo la gastrulación, la neuralización y la formación de placodas hasta llegar a las diferentesestructuras que conforman el sistema nervioso central y los órganos de los sentidos...

The nervous system has its origin in the well-known ectodermic germinal layer. During thepostnatal life, the nervous system is clearly differentiated in central nervous system andperipheral nervous system. During the embryonic stage, all the components follow differentpaths but they are still close to each other. This review focuses on the central nervoussystem and development of the sense organs. It includes a brief review of processes suchas gastrulation, neurulation and development of cranial placodes and the structures thatmake up the central nervous system and the sense organs...

The Effects of Retinoic Acid on Gastrulation of Hatched Mouse Embroyos / 대한산부인과학회잡지

Retinoic acid(RA), formed in vivo by oxidation of retinol, is known as morphogenic signal. RA plays an active role in normal embryonic development at physiological concentration, but excess RA can be a powerful teratogen in human and animals. The present study was designed to examine the direct effect of RA on murine embryogenesis(gastrulation) and to define the specific development processes perturbed by RA. Five to fifteen blastocysts were randomly assigned to separate culture dishes of the experimental group. Various concentrations of RA(10(-9) M, 10(-7) M, and 10(-5) M) were used in culturing blastocysts. In the effect of RA on the normal grouwth of embryo, the rates of development to the stages of attachment, early egg cylinder(EEC), late egg cylinder(LEC), and early somite(ES)were significantly(p < 0.01) decreased as the RA concentration increased. Stil in the yolk sac formation rate, there was a significant, dose-dependent difference(p < 0.01) according to the RA concentration. In the degeneration of embryos by RA, the effect was more apparent as the concentration of Ra increased. The production rates of embryos devoid of egg cylinder region and embryos with abnormal egg cylinder region were increased (p < 0.01)in a dose-dependent manner according to RA concentration. In conclusion, RA probably act as teratogen at gastrula stage embryos in high concentration and effect of teratogenesis is dose-dependent.