Wnt signaling pathway is involved in differentiation of embryonic stem cells into thymic epithelial progenitor cells together with autoimmune regulators / 中国组织工程研究

BACKGROUND:Autoimmune regulator gene(Aire)and Wnt signaling pathway play an important role in the maintenance and differentiation of mouse embryonic stem cell pluripotency.However,whether the Wnt signal and Aire are involved in the differentiation of embryonic stem cells to thymic epithelial progenitor cells remains poorly understood. OBJECTIVE:To investigate the relationship of the Wnt signaling pathway and Aire with the differentiation of embryonic stem cells. METHODS:A two-step differentiation method was used to induce mouse embryonic stem cells to differentiate into endoderm and then into thymic epithelial progenitor cells.Mouse embryonic stem cells were infected with Aire shRNA lentivirus,and monoclonal stable strains were screened by puromycin.Mouse embryonic stem cells were collected on days 0,3 and 10 of the directed induction of differentiation after the induced differentiation by the two-step differentiation method.Cellular immunofluorescence,flow cytometry,western blot assay,and real-time qPCR were used to detect the expression changes of related genes and proteins. RESULTS AND CONCLUSION:(1)Immunofluorescence staining showed positive expression of SSEA1 and OCT4 on day 0 of targeted induction of differentiation.(2)Immunofluorescence staining showed double-positive expression of SOX17 and FOXA2 on day 3 of targeted induction of differentiation.(3)Flow cytometry results showed positive expression of EPCAM1,K5 and K8 on day 10 of targeted induction of differentiation.(4)Compared with undifferentiated mouse embryonic stem cells,the expressions of Wnt7a,β-catenin,and Gsk-3β proteins were elevated,and the expression level of Aire protein was decreased in induced differentiated thymic epithelial progenitor cells.(5)Compared with undifferentiated mouse embryonic stem cells,the expressions of Wnt7a,β-catenin,Gsk-3β and Aire mRNA were elevated in thymic epithelial progenitor cells.(6)Compared with normal cultured mouse embryonic stem cells and their ultimately differentiated thymic epithelial progenitor cells,the expression levels of Wnt7a,β-catenin and Gsk-3β proteins were reduced in mouse embryonic stem cells with knockdown of Aire genes and their final differentiated thymic epithelial progenitor cells.In conclusion,the Wnt signaling pathway and Aire are jointly involved in the process of targeted induction of differentiation of mouse embryonic stem cells into mouse thymic epithelial progenitor cells.

DANCR Regulates hESC Differentiation Towards Definitive Endoderm / 中山大学学报(医学科学版)

ObjectiveTo explore the function of DANCR during the differentiation of human embryonic stem cells （hESC） toward definitive endoderm （DE）. MethodsThe in vitro DE differentiation system was established and its efficiency was verified. The correlation between the expression level of DANCR and DE differentiation process was detected. Using lentivirus system， we stably knocked down DANCR in hESC. The shDANCR hESC line was applied to DE differentiation， using qPCR and Western blot to detect the expression of DE marker genes SOX17 and FOXA2， and that of primitive streak marker genes Brachyury （T）， EOMES， MIXL1 and GSC. Dual luciferase reporter assay and qPCR were used to confirm the interaction between DANCR and the WNT pathway during DE differentiation. ResultsThe in vitro differentiation system mimicked DE differentiation efficiently. And the expression of DANCR was gradually downregulated during differentiation. DANCR was efficiently knocked down in the shDANCR hESC line （P < 0.001）. Compared with those in the control group， the expression levels of primitive markers Brachyury （T）， EOMES， MIXL1 and GSC， as well as DE markers SOX17 and FOXA2， were significantly decreased in shDANCR groups （P < 0.05）. Furthermore， the transcriptional activity of the WNT pathway in shDANCR groups was lower than that in the control group （P < 0.05）. And RNA levels of downstream genes of the WNT pathway， FZD5， FZD8， SFRP1， FRZB and ANKRD6， were significantly decreased in shDANCR groups （P < 0.05）. However， differences in protein levels of the TGFβ pathway effectors SMAD2/3 and p-SMAD2 were statistically insignificant in shDANCR and control groups （P > 0.05）. Forced activation of β-CATENIN rescued DANCR knock down-induced deficiency in DE differentiation. ConclusionsThe expression of DANCR decreases during DE differentiation. DANCR may promote DE differentiation through modulating the activity of the WNT pathway.

Bilineage embryo-like structure from EPS cells can produce live mice with tetraploid trophectoderm

Self-organized blastoids from extended pluripotent stem (EPS) cells possess enormous potential for investigating postimplantation embryo development and related diseases. However, the limited ability of postimplantation development of EPS-blastoids hinders its further application. In this study, single-cell transcriptomic analysis indicated that the "trophectoderm (TE)-like structure" of EPS-blastoids was primarily composed of primitive endoderm (PrE)-related cells instead of TE-related cells. We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure. Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation. Furthermore, we demonstrated that blastocyst-like structures reconstituted by combining the EPS-derived bilineage embryo-like structure (BLES) with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses. In summary, our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.

Uso del lexema δερµα (derma) en terminologia embryologica / Use of the lexeme δερµα (derm) in terminologia embryologica

Al lexema δερµα (derma) que proviene de las raíces griegas δέρ-µα/µατος se lo define como piel, pellejo, cuero, odre (Cortez, 2011). Lo encontramos en los términos ectodermo, mesodermo y endodermo, utilizados para describir las estructuras durante la tercera semana del desarrollo embriológico humano. Se consultó el significado y sus raíces en el diccionario Manuel Griego clásico-Español Vox (Pabón, 1967) y Diccionario Médico-Biológico, Histórico y Etimológico (DICCIOMED) de la Universidad de Salamanca (Cortez); de igual manera se investigó la utilización de los términos ectodermo, mesodermo y endodermo en la Terminologia Embryologica (FIPAT, 2013) y en su última versión (FIPAT, 2017). La búsqueda reportó que estos términos están compuestos por dos raíces griegas el sufijo δέρµα (derma) presente en los tres términos; más los prefijos ἐκτός que significa externo; µÎ­σος definido como medio y ἐνδο cuyo significado es dentro. Estos tres tejidos se derivan a la vez del epiblasto que viene de dos raíces griegas ἐπί- ep(í) que significa sobre + ßλαστός - blast(o) que se traduce como germen, retoño, forma celular inmadura; y del hipoblasto que cuyo término se forma de las raíces griegas ὑπό (hypó) que significa 'debajo de' + ßλαστός - blast(o). Podemos señalar que el mejor término para denominar a estas tres estructuras debiera ser ßλαστός (blasto); y por lo tanto, se deberían denominar a estas tres estructuras como ectoblasto, mesoblasto y endoblasto; debido a que son células o tejidos inmaduros, transitorios y no tejidos definitivos como es la piel; lo cual a su vez se corresponde con los objetivos determinados por la FIPAT.

The lexeme δέρ-µα (derma) that comes from the Greek δέρ-µα/µατος is defined as skin, hide, leather, wineskin (Cortez, 2011). We find it in the term ectoderm, mesoderm and endoderm, used to describe the structures during the third week of human embryological development. The meaning and its roots were consulted in the Manuel Greek-Spanish Vox dictionary (Pabón, 1967) and Medical-Biological, Historical and Etymological Dictionary (DICCIOMED) of the University of Salamanca (Cortez); the same way, the use of the terms ectoderm, mesoderm and endoderm was investigated in Terminologia Embryologica (FIPAT, 2013) and in its latest version Terminologia Embryologica (FIPAT, 2017). The search reported that these terms are composed of two Greek roots, the suffix δέρµα (derma) present in the three terms; plus the prefixes ἐκτός which means external; µÎ­σος defined as medium and ἐνδο whose meaning is within. These three tissues are derived in turn from the epiblast that comes from two Greek roots ἐπί- ep (í) which means over + ßλαστός - blast (o) which translates as germ, shoot, immature cell form; and from the hypoblast whose term is formed from the Greek roots ὑπό (hypó) meaning 'under' + ßλαστός - blast (o). We can say that the best term to name these three structures should be ßλαστός (blast); and therefore, these three structures should be named as ectoblast, mesoblast and endoblast; because they are immature, transitory cells or tissues and definitive non-tissues such as the skin; which in turn corresponds to the objectives determined by FIPAT.

Morphological Variations in Fissures and Lobar Pattern in Human Lungs.

Background: Our study aims to study the variations in lobar pattern and fissures of both right and left human lungs. Methods: 41 formalin fixed cadaveric lungs were obtained from the Department of Anatomy, SRMS IMS. The lungs were studied to observe the variations of fissures and lobes. Abnormal or accessory lobes were also noted. Results: 18 right lung and 23 left lung specimens were obtained and studied. Among the right lungs studied one showed an incomplete oblique fissure, six showed incomplete horizontal fissures. The horizontal fissure was absent in three right lungs. In the left lung only one lung showed the presence of an incomplete oblique fissure. Conclusion: The lung is a vital organ for life. Hence, considering the clinical importance of such anomalies, we as anatomists suggest that awareness and knowledge of the variations in the lobes and fissures of the lungs may be important for surgeons planning lobectomies and surgical resections involving individual segments and for radiologists to accurately interpret radiological images. This knowledge has further become more significant with the increasing incidence of lung carcinomas.

Promoting effects of activated Nodal signal pathway on definitive endo-derm induction from mouse embryonic stem cells / 中国病理生理杂志

[ ABSTRACT] AIM:To study the process of promoting mouse embryonic stem cells ( ESC) to specify to definitive endoderm by up-regulating of Nodal signal pathway in order to find the best cultivated systems of differentiation of mouse ESC to definitive endoderm cells.METHODS:The cells were divided into different groups based on the culture medium:ESC group ( serum-free medium +LIF) , natural differentiation group ( serum-free medium) and activin A group ( serum-free medium +50μg/L activin A).The cells and the sterilized coverslips with cells were collected at 1, 3, 5 and 7 d of the cultivation.The proportion of CXCR4 +cells was detected by flow cytometry.The expression of CXCR4 was determined by immunocytochemical method, and the protein expression of OCT4 and CXCR4 was detected by Western blot.RE-SULTS:The proportion of CXCR4 +cells showed no dramatic change in ESC group along with the extending of cultivation day, while there were gradually increased in natural differentiation group and activin A group and the highest level was ob-served at 5 d.Among the 3 groups, the proportion of CXCR4 +cells at 5 d was the highest in activin A group.The brown or tan staining in the cells observed under microscope was considered as positive CXCR4 by immunocytochemistry.The pro-tein levels of OCT4 and CXCR4 in ESC group along with the extending of cultivation days was observed.The expression levels of OCT4 were gradually decreased in the cells in natural differentiation group and activin A group, while those of CX-CR4 were gradually increased with the highest level at 5 d.It was highest in the cells in activin A group.CONCLUSION:The proportion of definitive endoderm was the highest at 5 d of the induction during in vitro mouse ESC differentiation.Up-regulation of Nodal signal pathway by adding activin A at the early stage of mouse ESC differentiation promotes mouse ESC to specify to definitive endoderm with CXCR4 molecular marker.

Dinâmica da inversão do saco vitelino em preás (Galea spixii Wagler, 1831) / Dynamics of yolk sac inversion in galea (Galea spixii Wagler, 1831)

O objetivo deste trabalho foi estudar o período de inversão do saco vitelino bem como a dinâmica resultante deste processo na gestação inicial em preás, utilizando-se microscopia de luz, microscopia eletrônica de varredura e de transmissão. No décimo segundo dia de gestação observou-se o desenvolvimento dos endodermas parietal e visceral delimitando a cavidade do saco vitelino. O endoderma parietal foi evidenciado revestindo a superfície fetal da placenta corioalantoidea bem como contornando o espaço delimitado pela decídua capsular. Estes endodermas apresentaram formato prismático e encontraram-se separados do trofoblasto por uma desenvolvida membrana de Reichert. Já o endoderma visceral continha vasos vitelínicos e possuía vilosidades apenas em determinadas áreas. No décimo quarto dia de gestação verificou-se a inversão do saco vitelino, caracterizada pela degeneração do endoderma parietal e trofoblasto mural, associado ao desaparecimento gradual da membrana de Reichert. Como consequência deste fenômeno, o endoderma visceral passou a constituir uma interface com o epitélio uterino. Após a inversão, o endoderma parietal que permaneceu íntegro foi aquele que se apoiava na superfície da placenta corioalantóidea, apresentando células em formato colunar alto e característica de epitélio pseudoestratificado. O endoderma visceral apresentou numerosas vilosidades apicais principalmente em regiões próximas a placenta corioalantóidea. Com o contínuo desenvolvimento do embrião e placenta corioalantóidea, observou-se o surgimento de importante área de aposição entre os endodermas visceral e parietal. A inversão do saco vitelino representou uma disposição anatômica favorável ao desenvolvimento embrionário, além de ser uma característica evolutiva nesta espécie de roedor.

The aim of this study was to study the time of yolk sac inversion as well as the dynamics resulting from this process in galea throughout pregnancy. For this, conventional histological techniques, scanning electron microscopy and transmission electron microscopy were used. Parietal and visceral endoderm delimiting the yolk sac cavity was observed at 12 days of pregnancy. The parietal endoderm was coating the fetal surface of the chorioallantoic placenta as well as delimiting the decidua capsularis area. This endoderm had prismatic format and were apart from the trophoblast by an enlarged Reichert's membrane. The visceral endoderm had vitelline vessels and there were villi only in certain areas. At 14 days of pregnancy the yolk sac inversion was characterized by the degeneration of parietal endoderm and mural trophoblast, and also the gradual disappearance of the Reichert's membrane. So it made the visceral endoderm establish an interface with the uterine epithelium. After the inversion, the parietal endoderm which remained intact was the one that rested on the chorioallantoic placenta surface. It presented cells with high columnar format and pseudostratified epithelium featured. The visceral endoderm presented many apical villi, especially in areas close to the chorioallantoic placenta. The continued development of the embryo and chorioallantoic placenta evidenced the emergence of an important apposition area between visceral and parietal endoderm. The yolk sac inversion represented an anatomical arrangement in favor of the embryo development as well as an evolutionary trait in this rodent species.

Sonic hedgehog inhibition induces mouse embryonic stem cells to differentiate toward definitive endoderm.

In the experimental group (shh inhibited group), there were significant decreases in the expression of Oct4, Nanog, Shh, GATA4, Brachyury and Goosecoid, while increases were observed for TAT and Pdx1. The expression of Sox17 did not differ between two control and experimental groups. In experimental group, the amount of GSC positive cells was somehow lower but it seems that there was no difference for Sox17. Shh inhibition induces ESCs to differentiate toward definitive endoderm by committing mesendodermal lineages.

Serum and non-serum method-induced differentiation of mouse embryonic stem cells into definitive endoderm / 第二军医大学学报

Objective: To compare the efficiencies of two methods (serum and non-serum) in inducing mouse embryonic stem cell differentiation into definitive endoderm cells. Methods: The serum and non-serum methods were used to induce differentiation of mouse embryonic stem cells into definitive endoderm cells. Fluorescence activated cell sorter (FACS) was used to analyze the inducing time and efficiency of definitive endoderm using their surface protein marks (Cxcr4, c-Kit and E-cadherin). Meanwhile, RT-PCR was used to analyze the gene profile of definitive endoderm induced by the two methods. Realtime PCR was used to analyze the gene expression in definitive endoderm during the induction course in the non-serum group. The Cxcr4 and c-Kit double positive definitive endoderm cells were sorted by flow cytometry and gene profile was characterized by RT-PCR. Results: Definitive endoderm cells were induced from mouse embryonic stem cells by both serum and non-serum methods. However, the efficiency of non-serum group (74. 19%) was higher than that in the serum group, and the induction outcome reached a climax at the 4th day of induction. Conclusion: We have established a highly efficient method to induce differentiation of mouse embryonic stem cells into definitive endoderm, which lays a foundation for further differentiation into liver and pancreatic cells.

Desarrollo del Aparato Digestivo / Digestive System Development

El aparato digestivo deriva del endodermo y el mesodermo, que forman su epitelio y la musculatura lisa respectivamente. Al igual que en el resto de los sistemas, existe un interacción epitelio-mesenquimática mediada por moléculas como Hedgehog, BMP y FoxF1 que determinan el crecimiento intestinal en sus ejes principales. Los genes Hox, junto con el resto de las moléculas, participan en la regionalización del sistema digestivo. En sus inicios lo denominaremos intestino primitivo, formado por un tubo endodérmico que deriva del saco vitelino; dividiéndose en intestino anterior, medio y posterior. En esta revisión veremos cómo estos 3 segmentos darán origen a las diferentes estructuras del sistema digestivo en los vertebrados.

The digestive system is derived from the endoderm and mesoderm, which form its epithelium and smooth muscle, respectively. As in the other systems, there is an epithelial-mesenchymal interactions mediated by molecules such as Hedgehog, BMP and FoxF1, determining intestinal growth in the main axes. The Hox genes, together the rest of the molecules, involved in the regionalization of the digestive system. In the beginning we call it primitive gut, consisting of a tube derived of endodermal yolk sac, divided into foregut, midgut and hindgut. In this review we will see how these 3 segments give rise to different structures of the digestive system in vertebrates.

Differentiation of human labia minora dermis-derived fibroblasts into insulin-producing cells

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin-producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis-derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal-endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic beta-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin-induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.

Neo-regeneration of urinary bladder: A desired metaplasia of autologous membrane from rectosigmoid colon containing stem cells of intestinal crypts.

The current management of diseases of urinary bladder requiring resection is by augmentation cystoplasty or transplantation of ureters. Transplantation of ureters is associated with morbidity and mortality. Ideal management will be by regenerating urinary bladder in vivo. Neo-regeneration of tissues and organs like abdominal wall, aponeurosis etc., has been attempted and patented. After neo-regeneration of mesoderm tissues and organs, regeneration of urinary bladder (developed from endoderm) was. In vivo surgical techniques were developed in dogs. It is known that the embryonic morphogenesis of urinary bladder is from uro-genital sinus of hind gut. A membrane, containing endoderm stem cells in crypts of recto-sigmoid colon, was surgically isolated and colonized with remnant of urinary bladder wall after extensive resection. Experimental study was performed in dogs, for 60 days to one and a half year. Regeneration of all the layers of tissues of the wall of urinary bladder was observed. The neo-regeneration phenomenon has been recognized as “desired metaplasia”. The regenerated neo tissue/organ on histological examination and cystometry studies was found compatible with normal urinary bladder. The hypothesis, neo-regeneration and desired metaplasia, is discussed.

The origin of endoderm: Transmission electron microscopic point of view.

Objective: To show the transmission electron microscopic (TEM) evidence to confirm that the endoderm originates from the epiblast of the primitive streak or from other sources. Methods: 60 fertilized Leghorn hen’s eggs were used in this study by incubating the eggs for about 18-27 hours at 38oC, then the chick embryos of the primitive streak stage to 7-somite stage were further processed for routine TEM study at the region of the primitive streak. Results: The epiblast proliferates and accumulates to form the primitive streak at the midcaudal of the embryonic disc from 18-27 hours incubation which corresponds with the early third week of the human embryo. TEM evidence shows that the epiblast at the primitive streak is the stratified columnar type of epithelium while the hypoblast is the simple squamous and the mesoderm cells are irregular in shape. The process of gastrulation begins with the formation of the filopodia of the epiblast by numerous protrusions of the plasma membrane from lateral side of the cell. These structures initiate the separation of the contacted cells. The deepest epiblast cells separate first while the superficial epiblast cells exhibit the desmosome between the adjacent cells. The separated epiblast cells are bottle-shaped with numerous filopodia and gradually change the shape into round or oval cells which migrate in the space between the epiblast and hypoblast. Some of these migrate to the hypoblast and contact with the hypoblast, the mesoblasts lose the filopodia and gain more close contact to the hypoblasts which become a very thin sheet of cells. The facing cell membrane later gradually disappears and the mesoblast then occupies the region of pre-existing hypoblast. There is no evidence that the mesoblast displaces the pre-existing hypoblast laterally to form the extraembryonic endoderm. Conclusion: These are TEM evidences that the epiblast of the primitive streak separates and migrates to form the mesoblast and some contact with the hypoblast. The later process appeared to reveal that the mesoblast compresses the hypoblast until the facing plasma membrane disappears and occupies the region of the pre-existing hypoblast.

Mesenchymal Stem Cells: The Promotion of Endodermal-Induction Using Activin A

PURPOSE: The most important consideration for therapy using MSCs is the differentiation of the target organ's cell type. For in-vitro hepatogenic differentiation of MSCs, the main focus is efficient induction of the MSCs into the endoderm stage. Activin A, which is a signaling molecule that is similar to Nodal, promotes the induction of definitive endoderm from both ESs and MSCs. The protocols for induction into definitive endoderm have shown different efficiency and reproducibility depending on the researchers or the sources of the MSCs. Thus, a study on the various conditions of Activin A is needed to efficiently differentiate MSCs into the definitive endoderm lineage of MSCs. METHODS: MSCs were isolated from human adipose tissues and these were cultured in MCM (MSCs Culture Medium) on a human fibronectin coated plate. At 70~80% confluence, the MSCs were harvested and cultured in MCM supplemented with Activin A, at a 50 ng/mL concentration, and FGF4. The expression of the genes related with MSCs or primitive endoderm were analyzed by RT-PCR. The changes of cell morphology for differentiation were also observed by a light microscope & a SEM. RESULTS: The expression of genes related with primitive foregut endoderm was seen in the groups that were treated with a higher concentration of Activin A. The morphology of the cells that differentiated into definitive endoderm were not different from those of the undifferentiated MSCs. The expression of genes related with functional primitive hepatocytes was seen in the early phase during hepatic differentiation. The cell morphology was changed to a similar cuboidal form in a time-dependent manner. CONCLUSION: Activin A promotes a more rapid induction of definitive endoderm. It also makes an efficient condition for the differentiation into primitive foregut endoderm at a higher concentration.

Mesenchymal Stem Cells: The Promotion of Endodermal-Induction Using Activin A

PURPOSE: The most important consideration for therapy using MSCs is the differentiation of the target organ's cell type. For in-vitro hepatogenic differentiation of MSCs, the main focus is efficient induction of the MSCs into the endoderm stage. Activin A, which is a signaling molecule that is similar to Nodal, promotes the induction of definitive endoderm from both ESs and MSCs. The protocols for induction into definitive endoderm have shown different efficiency and reproducibility depending on the researchers or the sources of the MSCs. Thus, a study on the various conditions of Activin A is needed to efficiently differentiate MSCs into the definitive endoderm lineage of MSCs. METHODS: MSCs were isolated from human adipose tissues and these were cultured in MCM (MSCs Culture Medium) on a human fibronectin coated plate. At 70~80% confluence, the MSCs were harvested and cultured in MCM supplemented with Activin A, at a 50 ng/mL concentration, and FGF4. The expression of the genes related with MSCs or primitive endoderm were analyzed by RT-PCR. The changes of cell morphology for differentiation were also observed by a light microscope & a SEM. RESULTS: The expression of genes related with primitive foregut endoderm was seen in the groups that were treated with a higher concentration of Activin A. The morphology of the cells that differentiated into definitive endoderm were not different from those of the undifferentiated MSCs. The expression of genes related with functional primitive hepatocytes was seen in the early phase during hepatic differentiation. The cell morphology was changed to a similar cuboidal form in a time-dependent manner. CONCLUSION: Activin A promotes a more rapid induction of definitive endoderm. It also makes an efficient condition for the differentiation into primitive foregut endoderm at a higher concentration.

Spatiotemporal regulation of fibroblast growth factor signal blocking for endoderm formation in Xenopus laevis

We have previously shown that the inhibition of fibroblast growth factor (FGF) signaling induced endodermal gene expression in the animal cap and caused the expansion of the endodermal mass in Xenopus embryos. However, we still do not know whether or not the alteration of FGF signaling controls embryonic cell fate, or when FGF signal blocking is required for endoderm formation in Xenopus. Here, we show that FGF signal blocking in embryonic cells causes their descendants to move into the endodermal region and to express endodermal genes. It is also interesting that blocking FGF signaling between fertilization and embryonic stage 10.5 promotes endoderm formation, but persistent FGF signaling blocking after stage 10.5 restricts endoderm formation and differentiation.

In vitro Differentiation of Human Embryonic Stem Cells into Definitive Endodermal Cells / 대한이식학회지

PURPOSE: Whole liver transplantation has limitation including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients. However, hepatocytes have limitation in proliferation and lose their property during culture period. To over come these problems, here we performed differentiation of human embryonic stem cells (hESCs) into definitive endoderm in order to differentiate into hepatocytes efficiently. METHODS: Undifferentiated hESCs were maintained on mouse embryo fibroblast feeder (MEF) layer for 5~7 days. For endoderm differentiation, we used modified Kevin A D'Amour's method that added 100 ng/mL Activin A for 5 days. After differentiation, differentiated endodermal cells were collected and RT-PCR and immunostain analysis were performed. RESULTS: After 5 days of differentiation period, hES cells showed endoderm committed-cells and increased expression of endoderm-specific marker genes (Sox17 and Foxa2). Also differentiated endoderm cells were stained with Sox17 and Foxa2 whereas undifferentiated hES cells were not stained with Sox17, Foxa2. CONCLUSION: In vitro differentiotion from hES cells to definitive endoderm was done repetitively by our methods. Further well defined protocol for differentiation of definitive endoderm to hepatocytes should be made.

A Case of Median Raphe Cyst on the Scrotum with Atypical Histologic Features / 대한피부과학회지

Median raphe cyst is an uncommon condition occuring on the ventral median raphe from the glans penis to the anus and represents a defect in the embryologic development of the male genitalia. The cyst wall may derive from endoderm, ectoderm, or mucous glands which are a normal constitute of the male urethra. It is classified as either a dermoid cyst lined by stratified squamous epithelium of ectodermal origin or a mucous cyst lined by cuboidal or columnar epithelium of endodermal origin. Surgical excision is the treatment of choice. We report a case of an 11-year old boy with three median raphe cysts of the scrotum. The epithelial lining of the cysts was mainly composed of pseudostratified columnar cells with decapitation secretion and focally showed stratified squamous cells and a transitional zone of two types of cells. Immunohistochemically, pseudostratified columnar cells showed CK 7 and CK 13 positivity, but were negative for CK 20. Besides, stratified squamous cells were negative for CK 7, CK 13 and CK 20. We report a rare case of median raphe cyst that is a combined type of dermoid cyst and mucous cyst.

Temporal Inhibition of FGF Signal on Endoderm Formation during Early Xenopus laevis Embryogenesis / 대한해부학회지

Our previous results showed that FGF signaling, which is important for the mesoderm and neuroectoderm induction, should be blocked for the endoderm formation in Xenopus. Here, Xenopus embryos were collected according to the two time points of MBT or stage 10.5. FGF signal was blocked with SU5402, chemical inhibitor of FGF signal, in the stage-specific embryos, to understand the role of FGF signal during the endoderm formation in the stage-specific embryos. Embryos subjected with the blocking of FGF signal before stage 10.5 showed the expanded abdominal volume in which endodermal mass was increased about 2 times but abdominal organs were not found. The tissue recombinant experiment showed that mesodermal tissue was necessary for the differentiation of endoderm. Embryos subjected with the blocking of FGF signal after stage 10.5 showed that abdomen was not expanded, the neural tube was opened instead. Our data indicate that blocking of FGF signal before stage 10.5 may be necessary for the endoderm induction and signals from neighboring endoderm tissue and mesoderm are required for the endoderm differentiation.

Intracranial immature teratoma with syncytiotrophoblasts and tumor marker positive intestinal lining cells

Intracranial teratomas are rare entities that can present as a pure type or as mixed germ cell tumor. Cases of mixed germ cell tumor composed of immature teratoma and choriocarcinoma have been reported. Also, immature teratoma can be mixed with only syncytiotrophoblasts. We report a case of immature teratoma with syncytiotrophoblasts of the brain discovered in a 3-year-old male baby. Serum human chorionic gonadotrophin (hCG) was normal and serum alpha-fetoprotein (AFP) was elevated. The tumor was mainly composed of intestinal glands, and neither endodermal sinus tumor nor embryonal carcinomatous elements were found. The cells lining the intestinal glands were positive for hCG and AFP. These findings suggest that the syncytiotrophoblasts are differentiated from the endoderm and AFP is not necessarily a marker exclusive to endodermal sinus tumor or embryonal carcinoma.