A New Approach to Generate Gastruloids to Develop Anterior Neural Tissues.

Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering. Key features A new gastruloid culture system to model post-implantation mouse embryonic development in vitro High-throughput formation of epiblast-like aggregates on hydrogel microwells Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues Graphical overview.

Bioengineered embryoids mimic post-implantation development in vitro.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids, with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids. Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro.

Gastruloids generated without exogenous Wnt activation develop anterior neural tissues.

When stimulated with a pulse from an exogenous WNT pathway activator, small aggregates of mouse embryonic stem cells (ESCs) can undergo embryo-like axial morphogenesis and patterning along the three major body axes. However, these structures, called gastruloids, currently lack the anterior embryonic regions, such as those belonging to the brain. Here, we describe an approach to generate gastruloids that have a more complete antero-posterior development. We used hydrogel microwell arrays to promote the robust derivation of mouse ESCs into post-implantation epiblast-like (EPI) aggregates in a reproducible and scalable manner. These EPI aggregates break symmetry and axially elongate without external chemical stimulation. Inhibition of WNT signaling in early stages of development leads to the formation of gastruloids with anterior neural tissues. Thus, we provide a new tool to study the development of the mouse after implantation in vitro, especially the formation of anterior neural regions.

Capturing Cardiogenesis in Gastruloids.

Organoids are powerful models for studying tissue development, physiology, and disease. However, current culture systems disrupt the inductive tissue-tissue interactions needed for the complex morphogenetic processes of native organogenesis. Here, we show that mouse embryonic stem cells (mESCs) can be coaxed to robustly undergo fundamental steps of early heart organogenesis with an in-vivo-like spatiotemporal fidelity. These axially patterned embryonic organoids (gastruloids) mimic embryonic development and support the generation of cardiovascular progenitors, including first and second heart fields. The cardiac progenitors self-organize into an anterior domain reminiscent of a cardiac crescent before forming a beating cardiac tissue near a putative primitive gut-like tube, from which it is separated by an endocardial-like layer. These findings unveil the surprising morphogenetic potential of mESCs to execute key aspects of organogenesis through the coordinated development of multiple tissues. This platform could be an excellent tool for studying heart development in unprecedented detail and throughput.

Mammary epithelial morphogenesis in 3D combinatorial microenvironments.

Human mammary epithelial cells can proliferate and reorganize into polarized multi-cellular constructs in-vitro, thereby functioning as an important model system in recapitulating key steps of in-vivo morphogenesis. Current approaches to constructing such three-dimensional mimics of the in-vivo microenvironment have involved the use of complex and ill-defined naturally derived matrices, whose properties are difficult to manipulate independently, and which have therefore limited our ability to understand the extrinsic regulation of morphogenesis. Here, we employ an automated, high-throughput approach to array modular building blocks of synthetic components, and develop a systematic approach to analyze colonies resulting from these varied microenvironmental combinations. This methodology allows us to systematically map the relationship between microenvironmental properties and ensuing morphogenetic phenotypes. Our analysis reveals that apico-basal polarity of mammary epithelial cells occurs within a narrow range of matrix stiffness, and that phenotypic homogeneity is favored in matrices which are insensitive to MMP-mediated degradation. Furthermore, combinations of extracellular proteins in the matrix finely tune the morphology of the mammary colonies, suggesting that subtle disregulations of the microenvironment may play a significant role in pathological disease states. This approach, which leverages the combinatorial possibilities of modular synthetic artificial extracellular matrices with an automated technology platform, demonstrates how morphogenesis can be assessed systematically in 3D, and provides new insights into mammary epithelial multicellularity.

High-throughput stem cell-based phenotypic screening through microniches.

As the field of tissue engineering develops, methods for screening combinations of signals for their effects on stem cell behavior are needed. We introduce a microgel-based screening platform for testing combinations of in situ-generated proteins on stem cell fate in ultrahigh-throughput. Compartmentalizing individual sets of growth factors was addressed by encapsulating aggregates of stable recombinant cell lines secreting individual glycoproteins into microgels through an on-chip polymerization. When these 'microniches' are cultured with a cell type of interest, fluorescence reporters indicate positive niches that perform the desired function, and the underlying producer cell lines of these selected microniches are analyzed by barcoded RNA sequencing. The microniche-based screening work-flow was validated via a model system based on engineered mammalian cells expressing yellow fluorescent protein (YFP) upon anti-inflammatory cytokine interleukin 4 (IL4)-based activation.

Multi-axial self-organization properties of mouse embryonic stem cells into gastruloids.

The emergence of multiple axes is an essential element in the establishment of the mammalian body plan. This process takes place shortly after implantation of the embryo within the uterus and relies on the activity of gene regulatory networks that coordinate transcription in space and time. Whereas genetic approaches have revealed important aspects of these processes1, a mechanistic understanding is hampered by the poor experimental accessibility of early post-implantation stages. Here we show that small aggregates of mouse embryonic stem cells (ESCs), when stimulated to undergo gastrulation-like events and elongation in vitro, can organize a post-occipital pattern of neural, mesodermal and endodermal derivatives that mimic embryonic spatial and temporal gene expression. The establishment of the three major body axes in these 'gastruloids'2,3 suggests that the mechanisms involved are interdependent. Specifically, gastruloids display the hallmarks of axial gene regulatory systems as exemplified by the implementation of collinear Hox transcriptional patterns along an extending antero-posterior axis. These results reveal an unanticipated self-organizing capacity of aggregated ESCs and suggest that gastruloids could be used as a complementary system to study early developmental events in the mammalian embryo.

Anteroposterior polarity and elongation in the absence of extra-embryonic tissues and of spatially localised signalling in gastruloids: mammalian embryonic organoids.

The establishment of the anteroposterior (AP) axis is a crucial step during animal embryo development. In mammals, genetic studies have shown that this process relies on signals spatiotemporally deployed in the extra-embryonic tissues that locate the position of the head and the onset of gastrulation, marked by T/Brachyury (T/Bra) at the posterior of the embryo. Here, we use gastruloids, mESC-based organoids, as a model system with which to study this process. We find that gastruloids localise T/Bra expression to one end and undergo elongation similar to the posterior region of the embryo, suggesting that they develop an AP axis. This process relies on precisely timed interactions between Wnt/ß-catenin and Nodal signalling, whereas BMP signalling is dispensable. Additionally, polarised T/Bra expression occurs in the absence of extra-embryonic tissues or localised sources of signals. We suggest that the role of extra-embryonic tissues in the mammalian embryo might not be to induce the axes but to bias an intrinsic ability of the embryo to initially break symmetry. Furthermore, we suggest that Wnt signalling has a separable activity involved in the elongation of the axis.

Neural tube morphogenesis in synthetic 3D microenvironments.

Three-dimensional organoid constructs serve as increasingly widespread in vitro models for development and disease modeling. Current approaches to recreate morphogenetic processes in vitro rely on poorly controllable and ill-defined matrices, thereby largely overlooking the contribution of biochemical and biophysical extracellular matrix (ECM) factors in promoting multicellular growth and reorganization. Here, we show how defined synthetic matrices can be used to explore the role of the ECM in the development of complex 3D neuroepithelial cysts that recapitulate key steps in early neurogenesis. We demonstrate how key ECM parameters are involved in specifying cytoskeleton-mediated symmetry-breaking events that ultimately lead to neural tube-like patterning along the dorsal-ventral (DV) axis. Such synthetic materials serve as valuable tools for studying the discrete action of extrinsic factors in organogenesis, and allow for the discovery of relationships between cytoskeletal mechanobiology and morphogenesis.

Emergency department visits caused by adverse drug reactions: results of aTurkish university hospital.

BACKGROUND/AIM: We aimed to evaluate adverse drug reaction (ADR)-related emergency department (ED) visits in the ED of the Dokuz Eylül University Hospital prospectively. MATERIALS AND METHODS: Patients who were admitted to the ED during 1-week periods of four different seasons between July 2010 and April 2011 were enrolled. Demographics of patients, previous ADR history, clinical progress, and outcomes were recorded. Causality assessment was done according to World Health Organization Uppsala Monitoring Centre categories. ADRs were categorized as certain, probable, or possible. RESULTS: Patients who were on medications (26.5%, n = 1838) were evaluated for ADR-related ED admissions. ADRs accounted for 5.9% of cases (n = 108). The most frequently affected systems were the gastrointestinal (35.2%, n = 38), dermatological (23.1%, n = 25), and hematological (10.2%, n = 11) systems (7.4%, n = 8). The most common causes of ADRs were antiinfectives (31.6%, n = 33). Amoxicillin, Coumadin, and paracetamol were the most common medications that caused ADRs. CONCLUSION: Nearly 6% of the admissions were ADR-related. ADRs should always be considered when patients who are on medication are admitted to the ED. Multicenter epidemiologic studies are required to know the real rates of ADR cases in EDs in Turkey.

An investigation of the emergency medicine journals published in Turkey.

INTRODUCTION: There are many academic journals in Turkey and the world. Medical journals have a significant place among those publications. The aim of this study is to examine qualitatively and categorize the scientific studies of the two journals in Turkey. It also aims to contribute the related literature in the area. MATERIAL-METHOD: Academic journals of medical emergencies published in Turkey between January 1, 2003 and December 20, 2014 were investigated in detail. All the works in journals were categorized briefly as research articles, case presentations, review articles, and other works. Moreover, research articles were investigated as observational and experimental, and discussed according to the including topics. RESULTS: 943 scientific works in 86 issues were fully investigated. The total number was found to be 472 for research articles (50.1%), 242 for case presentations (25.7%), 108 for review articles (11.5%), and finally it was 12.8% for other works. Research articles included 450 observational (95.3%) and 22 experimental studies (4.7%). The key topics covered in research articles were the management and training of medical emergencies, trauma 96 (20.3%), toxicology 50 (10.6%), and gastrointestinal tract 36 (7.6%). CONCLUSION: Despite its relatively short history, medical emergencies have improved progressively in Turkey. The number of domestic research articles has demonstrated an increase over the years. However, extra efforts are needed in order to improve the quality of articles. The most common contents encountered in research articles were the management and training of medical emergencies, trauma and toxicology.

Novel biochemical marker for differential diagnosis of seizure: ischemia-modified albumin.

INTRODUCTION: Differential diagnosis of seizure is critical in patients presented to emergency department (ED) with altered mental status or loss of consciousness. Although electroencephalogram is important for the diagnosis of seizures, its use in EDs is limited. The level of ischemia-modified albumin (IMA) increases in conditions of ischemic distress such as acute coronary syndrome, pulmonary embolism, and mesenteric ischemia. No studies exist in literature regarding the increase of IMA levels parallel to increased seizure activity in adults. The aim of the study was to investigate the diagnostic value of IMA in adult patients presented to ED with seizures. METHODS: Forty patients presented to ED with seizure, and 40 control patients of similar age and sex as the study group were enrolled in this study. Initial and fourth-hour levels of IMA and albumin were measured. Groups were compared in terms of sociodemographic data and details regarding their seizures as well as initial and fourth-hour levels of IMA. RESULTS: Mean levels of IMA were 61.5 IU/mL and 18.5 IU/mL (P < .001) initially and 56.7 IU/mL and 15.4 IU/mL (P < .001) at the fourth hour; levels were higher in the study group compared with control group. Ischemia-modified albumin/albumin ratios in study and control groups were 1555.3 IU/g and 462.4 IU/g (P < .001) initially and 1431.4 IU/g and 383.6 IU/g (P < .001) at the fourth hour, respectively. CONCLUSION: Blood IMA level and IMA/albumin ratio significantly increase in adult patients who experienced seizures. Ischemia-modified albumin may be considered as a useful biomarker in the differential diagnosis of seizure.

Isolated complex renal cyst mimicking kidney mass.

Hydatidosis is an endemic parasitic disease in Mediterranean region, Middle east, Australia, parts of Africa, Latin America and Turkey. The cysts are mostly evident in the liver or lungs, while urinary tract involvement is uncommon, comprising only 2-4 % of all cases. Isolated renal involvement is extremely rare. Its diagnosis may be difficult because of nonspecific complaints and absence of pathognomonic laboratory findings except from hydatiduria. Although radiological studies have a more important role in the diagnosis, they cannot always show a specific sign or lesion for hydatid disease. Herein we present a rare case with isolated renal hydatid cyst mimicking a renal mass treated with right radical nephrectomy. Isolated renal cyst hydatid should be considered in differential diagnosis of both cystic and solid renal masses.

Atypical presentations of retroperitoneal giant schwannomas.

Schwannomas are usually benign rare tumors that originating from Schwann cells of peripheral nerve sheaths. Presentation is generally varied and changed in a non-specific range from abdominal mass, flank pain to incidental findings. Herein we report 2 cases of retroperitoneal giant schwannomas with different clinical presentations, of whom one presented with vague abdominal pain, palpable abdominal mass for 4 years, swelling and bilateral hydronephrosis that caused by giant abdominal mass; the other one presented with right flank pain, rectal hemorrhage and lower extremities edema. Two patients were treated by complete surgical excision of masses. The histological and immunohistochemical diagnosis was reported as benign schwannoma. Both of patients are doing well and had no recurrence in 9 years and 28 months follow-up, respectively.