Embryonic Layers Research Articles

Induction of multipotency in umbilical cord-derived mesenchymal stem cells cultivated under suspension conditions

Due to the limitations in the clinical application of embryonic stem cells (ESC) and induced pluripotent stem cells, mesenchymal stem cells (MSCs) are now much more interesting for cell-based therapy. Although MSCs have several advantages, they are not capable of differentiating to all three embryonic layers (three germ layers) without cultivation under specific induction media. Hence, improvement of MSCs for cell therapy purposes is under intensive study now. In this study, we isolated MSCs from umbilical cord tissue at the single-cell level, by treatment with trypsin, followed by cultivation under suspension conditions to form a colony. These colonies were trypsin resistant, capable of self-renewal differentiation to the three germ layers without any induction, and they were somewhat similar to ESC colonies. The cells were able to grow in both adherent and suspension culture conditions, expressed both the MSCs markers, especially CD105, and the multipotency markers, i.e., SSEA-3, and had a limited lifespan. The cells were expanded under simple culture conditions at the single-cell level and were homogenous. Further and complementary studies are required to understand how trypsin-tolerant mesenchymal stem cells are established. However, our study suggested non-embryonic resources for future cell-based therapy.

Ellis-Van Creveld syndrome: Report of two cases

Ellis–van Creveld (EVC) syndrome is an ectodermal and chondral dysplasia as three embryonic layers are involved. It is a form of achondroplastic dwarfi sm resulting from an autosomal recessive disorder. It is most prevalent in the Amish population occurring in 1/5,000 live births and the birth prevalence in non-Amish population are estimated to be 7/1,000,000. Multiple malformations of the ectoderm and mesoderm, including short ribs, polydactyly, growth retardation and congenital cardiac anomalies are noted. This rare condition is inherited due to gene mutations of the EVC1 and EVC2, located on chromosome 4p16 in head to head confi guration. The presence of multiple orodental fi ndings such as conical teeth, hypodontia, malocclusion and hypoplasia of the enamel makes this syndrome important for a dentist. This article attempts to highlight on two female cases of EVC syndrome who reported to our institution where rare dental fi ndings and skeletal features were diagnosed along with a review of the literature.

Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.

The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems.

Mature Cystic Renal Teratoma

Teratomas are rare germline tumors that originate from one or more embryonic germ cell layers. Teratoma of the kidney is extremely rare, and less than 30 cases of primary intrarenal teratomas have been published to date. We report the main radiologic features of an unusual case of mature cystic teratoma arising from the left kidney in a two-year-old boy. A left-sided abdominal mass was detected on physical examination and B-Mod Ultrasound (US) examination revealed a heterogeneous mass with central cystic component. Computed tomography (CT) demonstrated a lobulated, heterogeneous, hypodense mass extending craniocaudally from the splenic hilum to the level of the left iliac fossa. Nephrectomy was performed and a large, fatty mass arising from the left kidney was excised. The final pathologic diagnosis was confirmed as cystic renal teratoma.

Macroscopic and microscopic study of the embryonic development of the desert locust Schistocerca gregaria (Forskal, 1775) (Orthoptera: Acrididae) in laboratory

Schistocerca gregaria (Forskal, 1775) is a serious pest of crops. The knowledge of its biology can contribute to the integrated pest management against locusts. In laboratory conditions (32 ± 2.9 °C and 70 ± 5.3% RH), the duration of the embryonic development of Schistocerca gregaria is 12.63 ± 0.55 days. The macroscopic study showed that the bright yellow egg at laying, changes to orange 2-3 hours after laying; then to Brown 1-2 days later and eventually becomes clear at the end of development. These color changes are accompanied by weight gain of 8.3-20 mg at the end of development. Observation by transparency of rudiments of organs was made from the 5th day. These transformations correspond to the stages of segmentation, gastrulation and organogenesis. These three stages interfere and overlap.Histological investigations reveal partial and superficial segmentation, characterized by an early Karyiokinesis followed by cytokinesis. After one hour and thirty minutes the periblastula is formed. Gastrulation is initiated immediately, with movements of cell proliferation, deployment, embolism and delamination leading to the formation of the embryonic layers within 24 hours. Clusters of cells from which will derive rudiments of organs are observed on sections of eggs of two days. Morphogenesis begins with cephalization with the formation of the optic lobes, then the thorax and abdomen. The whole morphogenesis lasts about ten days. Knowledge of the duration and timing of embryonic development will allow to provide the best times and the means of intervention to contribute to the integrated pest management (IPM) against locusts.

Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken

Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis.

Polygene-modified mouse induced differentiation of pluripotent stem cells into insulin-producing cells

Objective To evaluate the effects of key insulin gene transcription regulators(PDX-1,NeuroD1 and MafA) on the differentiation of induced pluripotent stem cells(iPSCs) into insulin-producing cells.MethodsMouse embryonic fibroblasts(MEFs) were infected with lentivirus(LV-ef1a-Hygromicin-TRE-Oct4/Sox2/Klf4/cMyc) at a multiplicity of infection,and iPSCs were selected and identified.Then the iPSCs were infected with adenovirus(Ad-mPDX-1-IRES-GFP,Ad-mNeuroD1-IRES-GFP and Ad-mMafA-IRES-GFP) to induce differentiation into insulin-producing cells in vitro.RT-PCR was applied to detect expression of functional genes in pancreatic islet B cells;immunofluorescence was used to examine the expression and location of insulin protein;and ELISA was used to determine the volumes of secreted insulin at different concentrations of glucose(0,5,10,20,30,and 40 mmol/L).ResultsThe iPSCs derived from MEFs could form intensive clones with smooth boundary,express embryonic stem cell-specific cell surface markers,including Nanog,SSEA-1 and Rex-1,and differentiate into three embryonic layers,which indicating that MEFs were successfully reprogrammed into iPSCs.Mouse iPSCs infcted with Ad-PDX-1-IRES-GFP,Ad-mNeuroD-IRES-GFP,and Ad-mMafA-IRES-GFP could differentiate into pancreatic islet B cells.RT-PCR results showed that polygene-modified iPSCs and pancreatic islet B-cell line MIN6 had similar gene expression profile.Immunofluorescence analyses confirmed insulin expression in the differentiated cells.Results of ELISA showed that polygene-modified iPSCs had a satisfactory response to different concentrations of glucose.ConclusionKey insulin gene transcription regulators PDX-1,NeuroD1 and MafA can work synergistically to induce mouse iPSCs differentiation into pancreatic islet B cells capable of insulin biosynthesis and secretion.

Stem Cells of Epidermis: A Critical Introduction

Stem cells are the cells having some distinguishing characteristics like longevity, high capacity of self-renewal and differentiation, quiescence and highly error-free proliferation. Almost all stem cells have the potential of lineage reprogramming, i.e. inter-conversion of cell lineages. They also have the potential to differentiate into almost all kinds of cells. These cells have been found in almost every organs of human body. Pool of stem cells found in epidermis is termed as Epidermal Stem Cells. Many researchers around the world have reported different kinds of stem cells in skin, based on their cell surface makers, while they have not categorized these cells chronically. It is worth mentioning here that all kinds of stem cells reported in skin, i.e. keratinocyte stem cells, limbal stem cells, hair follicle and bulge stem cells, SG (sebaceous gland) stem cells, and spinous keratinocytes express specific types of cytokeratin protein (e.g. K1, K3, K5, K10, K12, K14, K15, K19 etc.) on their surfaces. Biologists have defined that almost all of these stem cells share single origin, i.e. Basal Layer of Embryonic Skin. As the embryonic skin passes the developmental stages, the basal layer produces two mother stem cells of skin, keratinocyte stem cells, and so called Limbal Stem Cells (in cornea). Keratinocyte stem cells give rise to the cells expressing Cytokeratin proteins on their surfaces. So, all the cells expressing cytokeratin are tracked back to these stem cells in origin , while undergone natural lineage reprogramming or differentiation. The stem cells in the basal-layer give rise to keratinocyte stem cells which can be found in the basal layer of the adult skin. During the developmental stages, this basal layer, containing keratinocyte stem cells, gives rise to limbal invagination of corneal region, a linage conversion mechanism happens, and the keratinocyte stem cells are naturally reprogramed into the limbal stem cells. In basal layer of the epidermis, the keratinocyte stem cells give rise to bulge, hair follicle, and SG stem cells when placode formation takes place. In future, we need studies to find out which kind of cytokeratin protein is expressed early in these cells. In another word it would be helpful to define the order of cytokeratin expression regarding these lineage developmental processes from embryonic to mature skin and from embryonic basal layer to the formation of cornified epithelial cells.

ALK Family Inhibitor A83-01 Promotes the Proliferation of Mouse Male Germline Stem Cells (mGSCs) Under Serum- and Feeder-Free Conditions

A83-01 is a selective inhibitor of the TGF-β type I receptor ALK, which inhibits the TGF-β-induced epithelial-to-mesenchymal transition (EMT) via the inhibition of Smad2 phosphorylation. Previous studies have showed that A83-01 promoted somatic cellular reprogramming significantly. Male germline stem cells (mGSCs), as an alternative resource of pluripotent stem cells derived adult testis, have promising valuable in clinic medicine and regeneration, however, the derivation of mGSCs was complex and difficult. What the role A83-01 plays in promoting the proliferation of mGSCs is still unknown. In this study, combined with A83-01 and knockout serum replacement (KSR) medium, we obtained a relatively feeder- and serum-free system for mGSCs culturing in vitro and the optimal concentration of A83-01 was 0.25 μmol L−1. After continuous culturing, the proliferation efficiency of undifferentiated mGSCs and differentiation capacity of mGSC were examined as well. Results showed that, A83-01 dramatically increased the number of mGSCs and AP positive colonies, and the mitosis index according to the BrdU assay. A83-01 could also increase the expression of pluripotent markers including Oct4, Klf4, Nanog and c-Myc, analyzed by real-time quantative PCR. mGSCs cultured in the optimal feeder-and serum-free system combined with A83-01 could form embryoid bodies (EBs), which consisted of three embryonic layers detected by immunofluorescence and RT-PCR. Remarkably, the results demonstrated 0.25 μmol L−1 A83-01 could promote the proliferation of mouse mGSC colonies and maintain their undifferentiated status under feeder- and serum-free systems.

A comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ1–42, okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease.

A p38mapk-p53 cascade regulates mesodermal differentiation and neurogenesis of embryonic stem cells

Embryonic stem cells (ESCs) differentiate in vivo and in vitro into all cell lineages, and they have been proposed as cellular therapy for human diseases. However, the molecular mechanisms controlling ESC commitment toward specific lineages need to be specified. We previously found that the p38 mitogen-activated protein kinase (p38MAPK) pathway inhibits neurogenesis and is necessary to mesodermal formation during the critical first 5 days of mouse ESC commitment. This period corresponds to the expression of specific master genes that direct ESC into each of the three embryonic layers. By both chemical and genetic approaches, we found now that, during this phase, the p38MAPK pathway stabilizes the p53 protein level and that interfering directly with p53 mimics the effects of p38MAPK inhibition on ESC differentiation. Anti-p53 siRNA transient transfections stimulate Bcl2 and Pax6 gene expressions, leading to increased ESC neurogenesis compared with control transfections. Conversely, p53 downregulation leads to a strong inhibition of the mesodermal master genes Brachyury and Mesp1 affecting cardiomyogenesis and skeletal myogenesis of ESCs. Similar results were found with p53−/− ESCs compared with their wild-type counterparts. In addition, knockout p53 ESCs show impaired smooth muscle cell and adipocyte formation. Use of anti-Nanog siRNAs demonstrates that certain of these regulations result partially to p53-dependent repression of Nanog gene expression. In addition to its well-known role in DNA-damage response, apoptosis, cell cycle control and tumor suppression, p53 has also been involved in vivo in embryonic development; our results show now that p53 mediates, at least for a large part, the p38MAPK control of the early commitment of ESCs toward mesodermal and neural lineages.

Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line

PurposeMouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application.MethodsA novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2–3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells.ResultsESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation.ConclusionsHuman feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.

The (real) neurogenic/gliogenic potential of the postnatal and adult brain parenchyma.

During the last two decades basic research in neuroscience has remarkably expanded due to the discovery of neural stem cells (NSCs) and adult neurogenesis in the mammalian central nervous system (CNS). The existence of such unexpected plasticity triggered hopes for alternative approaches to brain repair, yet deeper investigation showed that constitutive mammalian neurogenesis is restricted to two small “neurogenic sites” hosting NSCs as remnants of embryonic germinal layers and subserving homeostatic roles in specific neural systems. The fact that in other classes of vertebrates adult neurogenesis is widespread in the CNS and useful for brain repair sometimes creates misunderstandings about the real reparative potential in mammals. Nevertheless, in the mammalian CNS parenchyma, which is commonly considered as “nonneurogenic,” some processes of gliogenesis and, to a lesser extent, neurogenesis also occur. This “parenchymal” cell genesis is highly heterogeneous as to the position, identity, and fate of the progenitors. In addition, even the regional outcomes are different. In this paper the heterogeneity of mammalian parenchymal neurogliogenesis will be addressed, also discussing the most common pitfalls and misunderstandings of this growing and promising research field.

Ellis Van Creveld Syndrome - A perinatal autopsy case report

Ellis Van Creveld syndrome is a disease complex where all the three embryonic layers appear to be involved. This disorder is also called chondro ectodermal dysplasia or meso ectodermal dysplasia. It is a part of an emerging class of diseases called ciliopathies due to primary dysfunction of cilia. It is very rare; only about 150 cases have been described in the world literature and rarely been described in the Indian literature. In our case perinatal autopsy was performed of a still born baby of 32 wks gestation having syndactyly and polydactyly and short forearm, to know the cause of death, and the diagnosis was emerged as Ellis Van Creveld syndrome. In India most of the intrauterine death cases remain undiagnosed, but diagnosis of EVC syndrome is important because it is transmitted as autosomal recessive manner so genetic counseling is required to make the parents aware of the risk of recurrence. Keywords - Ellis Van Creveld syndrome, perinatal autopsy, polydactyly I. INTRODUCTION Ellis Van Creveld syndrome is a rare genetic disorder of skeletal dysplasia type. The cardinal features usually present as tetrad of : a) disproportionate small stature with increasing severity from the proximal to distal portions of the limbs. b) Polydactyly, affecting hands and occasionally the feet. c) Hydrotic ectodermal dysplasia mainly affecting the nails, hair and teeth. d) Congenital heart malformations occurring in about 50%- 60% of cases and comprising of single atrium, defects of the mitral and tricuspid valves, patent ductus, ventricular septal defect, atrial septal defect and hypoplastic left heart syndrome. The presence of congenital heart disease may support the diagnosis of the EVC syndrome and appears to be the main determinant of longevity. (1) It is transmitted as autosomal recessive trait and caused by mutation of EVC gene ( both EVC1 and EVC2 ) and the locus is 4p16. (2) The largest pedigree of EVC syndrome has been described in the old order

Chitin-binding proteins of Artemia diapause cysts participate in formation of the embryonic cuticle layer of cyst shells

The brine shrimp Artemia reproduces either ovoviviparously, producing free-swimming nauplii, or oviparously, producing encysted embryos (diapause cysts) able to cope with harsh and complex habitats. When the cysts enter diapause they are encased in a complex external shell that protects them from certain extreme environments. The genomic comparison of oviparous and ovoviviparous ovisacs has been described previously. We isolated three significantly up-regulated genes in oviparous oocytes and identified them as Arp-CBP (Artemia parthenogenetica chitin-binding protein) genes. Quantitative real-time PCR indicated that the expression of Arp-CBP genes gradually increases during diapause cyst formation and significant mRNA accumulation occurs during the ovisac stage of oviparous development. Moreover, in situ hybridization results demonstrated that Arp-CBP mRNAs are expressed in the embryo. Interestingly, the results of immune electron microscopy showed that all three Arp-CBPs are distributed throughout the cellular ECL (embryonic cuticle layer) of the cyst shell. Furthermore, knockdown of Arp-CBP by RNA interference resulted in marked changes in the composition of the embryonic cuticular layer. The fibrous layer of the cyst shell adopted a loose conformation and the inner and outer cuticular membranes exhibited marked irregularities when Arp-CBP expression was suppressed. Finally, an invitro recombinant protein-binding assay showed that all three Arp-CBPs have carbohydrate-binding activities. These findings provide significant insight into the mechanisms by which the ECL of Artemia cyst shell is formed, and demonstrate that Arp-CBPs are involved in construction of the fibrous lattice and are required for formation of the ECL of the cyst shell.

308 Tet1 PLAYS IMPORTANT ROLES IN MAINTAINING CHARACTERIZATIONS OF PORCINE INDUCED PLURIPOTENT STEM CELLS

It was recently found that the ten-eleven-translocation (TET) family of Fe(II) and 2-oxoglutarate-dependent enzymes (Tet1/2/3) can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and thus promotes active demethylation of genomes. Tet1 is highly expressed in mouse embryonic stem cells (mESC) and has been demonstrated to involve in mESC maintenance. Here we used small interference RNA (siRNA) to transiently knockdown expression of Tet1 in porcine induced pluripotent stem cells (iPSC) in order to identify its functions. The fetal fibroblasts were isolated from a 30-day-old porcine fetus and induced into iPSC with defined transcription factors, namely Oct-4, Sox-2, Klf-4, and C-myc. The colonies appeared on Day 12 and were picked up on Day 14. These colonies had normal ES-like morphology and alkaline phosphatase activity. Specifically, they were positively stained for pluripotency-specific markers, including Oct4, Sox2, Nanog, Rex1, and SSEA1. When cultured in vitro, the cells formed embryoid bodies (EB), and all 3 germ layer markers (endoderm: AFP, alphaAT; mesoderm: BMP4, Enolase; ectoderm: GFAP, Neurod) were detected positively in EB. For siRNA transfections, iPSC from the colonies were transfected with 40 pmol of siRNA and 2 µL of Lipofectamine 2000 in 1 well of a 24-well plate. After transfection, iPSC were subjected to fluorescence-activated cell sorting to determine the fraction of FAM-positive cells in order to confirm transfection efficiency; the percentage of positive cells reached 48 ± 4.96. We observed obvious knockdown of Tet1 after short-term transfection of siRNA, and the knockdown efficiency was confirmed using qRT-PCR and immunofluorescence staining. Notably, knockdown of Tet1 resulted in morphological abnormality and loss of undifferentiated state of porcine iPSC. However, no obvious morphological changes were observed in the negative control (transfected with nonsense-siRNA), positive control (transfected with GAPDH-siRNA), or mock control (transfected with DEPC-treated water). To gain insight into the molecular mechanism underlying the self-renewal defect, we analysed the effects of Tet1 knockdown on the expression of key stem cell factors and differentiation markers of different embryonic layers using qRT-PCR. We found that knockdown of Tet1 resulted in downregulated expression of pluripotency-related genes, such as Lefty-2, Klf-2, and Sox-2 (the expression ratios of post-transfection to pre-transfection were 0.31 ± 0.21, 0.48 ± 0.072, and 0.65 ± 0.046, respectively), and upregulated expression of differentiation-related genes, including Pitx-2, Hand-1, Gata-6, and Lef-1 (the expression ratios of post-transfection to pre-transfection were 4.35 ± 1.36, 2.56 ± 0.68, 2.91 ± 1.47, and 2.33 ± 1.11, respectively). However, Oct-4, C-myc, Klf-4, and Nanog were not downregulated (the expression ratios of post-transfection to pre-transfection were 0.91 ± 0.15, 1.12 ± 0.26, 1.15 ± 0.21, and 1.08 ± 0.08, respectively). Taken together, Tet1 plays important roles in porcine iPSC self-renewal and characterization maintenance. This study was financed by National Basic Research Program of China (NO.2009CB941001).

Primary Ovarian Teratoma and GCT with Intra-abdominal Metastasis in a Dog

This report describes the simultaneous occurrence of an ovarian teratoma and a granulosa cell tumor (GCT) with intra-abdominal metastasis in a 1.5 yr old female Doberman pinscher. At surgery, a 20 cm, smooth, intact mass associated with the left ovary and multiple 1-2 cm irregular masses in the broad ligament were found. The masses were surgically removed and submitted for histopathology. A histologic diagnosis of a teratoma and a GCT with broad ligament metastasis was made. Further treatment was elected by the owner and included two cycles of carboplatin therapy. The dog was euthanized 6 wk postoperatively for signs related to metastasis and dyspnea. Teratoma of the ovary, although it contains derivatives of all three embryonic germ cell layers, rarely presents together with either ovarian epithelial or sex cord-stromal tumors. To the authors' knowledge, this is the first reported case of an ovarian teratoma coexisting with a primary GCT with intra-abdominal metastasis in the same ovary in a dog.

The ciliary proteins Meckelin and Jouberin are required for retinoic acid-dependent neural differentiation of mouse embryonic stem cells

The dysfunction of the primary cilium, a complex, evolutionarily conserved, organelle playing an important role in sensing and transducing cell signals, is the unifying pathogenetic mechanism of a growing number of diseases collectively termed “ciliopathies”, typically characterized by multiorgan involvement. Developmental defects of the central nervous system (CNS) characterize a subset of ciliopathies showing clinical and genetic overlap, such as Joubert syndrome (JS) and Meckel Syndrome (MS). Although several knock-out mice lacking a variety of ciliary proteins have shown the importance of primary cilia in the development of the brain and CNS-derived structures, developmental in vitro studies, extremely useful to unravel the role of primary cilia along the course of neural differentiation, are still missing. Mouse embryonic stem cells (mESCs) have been recently proven to mimic brain development, giving the unique opportunity to dissect the CNS differentiation process along its sequential steps. In the present study we show that mESCs express the ciliary proteins Meckelin and Jouberin in a developmentally-regulated manner, and that these proteins co-localize with acetylated tubulinlabeled cilia located at the outer embryonic layer. Further, mESCs differentiating along the neuronal lineage activate the cilia-dependent sonic hedgehog signaling machinery, which seems to be impaired in Meckelin knock-down cells but results unaffected in Jouberin-deficient mESCs. However, both seems to lose the ability to acquire a neuronal phenotype. Altogether these findings suggest a pivotal role of Meckelin and Jouberin during embryonic neural specification and indicate mESCs as a suitable tool to investigate the developmental impact of ciliary proteins dysfunction.

Culture conditions affect cardiac differentiation potential of human pluripotent stem cells.

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are capable of differentiating into any cell type in the human body and thus can be used in studies of early human development, as cell models for different diseases and eventually also in regenerative medicine applications. Since the first derivation of hESCs in 1998, a variety of culture conditions have been described for the undifferentiated growth of hPSCs. In this study, we cultured both hESCs and hiPSCs in three different culture conditions: on mouse embryonic fibroblast (MEF) and SNL feeder cell layers together with conventional stem cell culture medium containing knockout serum replacement and basic fibroblast growth factor (bFGF), as well as on a Matrigel matrix in mTeSR1 medium. hPSC lines were subjected to cardiac differentiation in mouse visceral endodermal-like (END-2) co-cultures and the cardiac differentiation efficiency was determined by counting both the beating areas and Troponin T positive cells, as well as studying the expression of OCT-3/4, mesodermal Brachyury T and NKX2.5 and endodermal SOX-17 at various time points during END-2 differentiation by q-RT-PCR analysis. The most efficient cardiac differentiation was observed with hPSCs cultured on MEF or SNL feeder cell layers in stem cell culture medium and the least efficient cardiac differentiation was observed on a Matrigel matrix in mTeSR1 medium. Further, hPSCs cultured on a Matrigel matrix in mTeSR1 medium were found to be more committed to neural lineage than hPSCs cultured on MEF or SNL feeder cell layers. In conclusion, culture conditions have a major impact on the propensity of the hPSCs to differentiate into a cardiac lineage.

Human decidua-derived mesenchymal stromal cells differentiate into hepatic-like cells and form functional three-dimensional structures

Previously, we have shown that human decidua-derived mesenchymal stromal cells (DMSC) are mesenchymal stromal cells (MSC) with a clonal differentiation capacity for the three embryonic layers. The endodermal capacity of DMSC was revealed by differentiation into pulmonary cells. In this study, we examined the hepatic differentiation of DMSC. DMSC were cultured in hepatic differentiation media or co-cultured with murine liver homogenate and analyzed with phenotypic, molecular and functional tests. DMSC in hepatic differentiation media changed their fibroblast morphology to a hepatocyte-like morphology and later formed a 3-dimensional (3-D) structure or hepatosphere. Moreover, the hepatocyte-like cells and the hepatospheres expressed liver-specific markers such as synthesis of albumin (ALB), hepatocyte growth factor receptor (HGFR), α-fetoprotein (AFP) and cytokeratin-18 (CK-18), and exhibited hepatic functions including glycogen storage capacity and indocyanine green (ICG) uptake/secretion. Human DMSC co-cultured with murine liver tissue homogenate in a non-contact in vitro system showed hepatic differentiation, as evidenced by expression of AFP and ALB genes. The switch in the expression of these two genes resembled liver development. Indeed, the decrease in AFP and increase in ALB expression throughout the co-culture were consistent with the expression pattern observed during normal liver organogenesis in the embryo. Interestingly, AFP and ALB expression was significantly higher when DMSC were co-cultured with injured liver tissue, indicating that DMSC respond differently under normal and pathologic micro-environmental conditions. In conclusion, DMSC-derived hepatospheres and DMSC co-cultured with liver homogenate could be suitable in vitro models for toxicologic, developmental and pre-clinical hepatic regeneration studies.