A High-Throughput Device for Patterned Differentiation of Embryoid Bodies

2013 ◽  
Vol 25 (4) ◽  
pp. 623-630 ◽  
Author(s):  
Xiaoming He ◽  
◽  
Hiroshi Kimura ◽  
Teruo Fujii ◽  
◽  
...  
Keyword(s):  
Microfluidic Device ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Culture Methods ◽  
Theoretical Simulation ◽  
Chemical Gradients ◽  
Conventional Culture ◽  
Differentiation And Proliferation

Although ontogenesis in vivo may proceed in a spatiotemporally heterogeneous environment, in vitro differentiation of an embryoid body (EB) has been carried out in uniform conditions using conventional culture methods at low throughput. In the present study, a microfluidic device with multiple culture chambers for simultaneous patterned differentiation of multiple EBs of pluripotent stem cells is newly developed. Theoretical simulation and experiments using a suspension of fluorescent particles or fluorescent solution show that proper chemical gradients can be formed with almost no flow in the chambers. After multiple EBs are introduced into the device, these EBs move along the flow channel and into trapping cups. The EBs are pushed by air bubbles into the culture chambers. These multiple EBs can be cultured within the culture chambers after flowing culture medium removes the air bubble from the device. In our experiment, differentiation and proliferation of these multiple EBs are studied by exposing them to two different media for 6 days: one to induce differentiation and the other to keep the pluripotent and self-renewing state of the cells. It is shown that patterned differentiation of the multiple EBs is successfully conducted simultaneously in the device when these two media are perfused into the device. The results suggest that differentiation and proliferation of multiple EBs can be analyzed by applying chemical gradients in the present microfluidic device. This will be a helpful tool in a wide variety of experiments involving EBs or spheroids.

scholarly journals Human Adipose-Derived Stromal/Stem Cell Culture and Analysis Methods for Adipose Tissue Modeling In Vitro: A Systematic Review

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1378
Author(s):  
Peyton Gibler ◽  
Jeffrey Gimble ◽  
Katie Hamel ◽  
Emma Rogers ◽  
Michael Henderson ◽  
...  
Keyword(s):  
Systematic Review ◽  
Adipose Tissue ◽  
Drug Discovery ◽  
3D Culture ◽  
Human Adipose Tissue ◽  
Culture Methods ◽  
Adipose Tissue Engineering ◽  
The Impact

Human adipose-derived stromal/stem cells (hASC) are widely used for in vitro modeling of physiologically relevant human adipose tissue. These models are useful for the development of tissue constructs for soft tissue regeneration and 3-dimensional (3D) microphysiological systems (MPS) for drug discovery. In this systematic review, we report on the current state of hASC culture and assessment methods for adipose tissue engineering using 3D MPS. Our search efforts resulted in the identification of 184 independent records, of which 27 were determined to be most relevant to the goals of the present review. Our results demonstrate a lack of consensus on methods for hASC culture and assessment for the production of physiologically relevant in vitro models of human adipose tissue. Few studies have assessed the impact of different 3D culture conditions on hASC adipogenesis. Additionally, there has been a limited use of assays for characterizing the functionality of adipose tissue in vitro. Results from this study suggest the need for more standardized culture methods and further analysis on in vitro tissue functionality. These will be necessary to validate the utility of 3D MPS as an in vitro model to reduce, refine, and replace in vivo experiments in the drug discovery regulatory process.

scholarly journals Prediction and control of symmetry breaking in embryoid bodies by environment and signal integration

2018 ◽  
Author(s):  
Naor Sagy ◽  
Shaked Slovin ◽  
Maya Allalouf ◽  
Maayan Pour ◽  
Gaya Savyon ◽  
...  
Keyword(s):  
Symmetry Breaking ◽  
Cell Fate ◽  
Developmental Process ◽  
Primitive Streak ◽  
Early Embryo ◽  
Embryoid Bodies ◽  
Neighboring Cell ◽  
Contact Points

AbstractDuring early embryogenesis, mechanical signals, localized biochemical signals and neighboring cell layers interaction coordinate around anteroposterior axis determination and symmetry breaking. Deciphering their relative roles, which are hard to tease apart in vivo, will enhance our understanding of how these processes are driven. In recent years, in vitro 3D models of early mammalian development, such as embryoid bodies (EBs) and gastruloids, were successful in mimicking various aspects of the early embryo, providing high throughput accessible systems for studying the basic rules shaping cell fate and morphology during embryogenesis. Using Brachyury (Bry), a primitive streak and mesendoderm marker in EBs, we study how contact, biochemical and neighboring cell cues affect the positioning of a primitive streak-like locus, determining the AP axis. We show that a Bry-competent layer must be formed in the EB before Bry expression initiates, and that Bry onset locus selection depends on contact points of the EB with its surrounding. We can maneuver Bry onset to occur at a specific locus, a few loci, or in an isotropic peripheral pattern. By spatially separating contact and biochemical signal sources, we show these two modalities can be integrated by the EB to generate a single Bry locus. Finally, we show Foxa2+ cells are predictive of the future location of Bry onset, demonstrating an earlier symmetry-breaking event. By delineating the temporal signaling pathway dependencies of Bry and Foxa2, we were able to selectively abolish either, or spatially decouple the two cell types during EB differentiation. These findings demonstrate multiple inputs integration during an early developmental process, and may prove valuable in directing in vitro differentiation.

scholarly journals Ratchetaxis in channels: cells move directionally by pushing walls asymmetrically

2020 ◽  
Author(s):  
Emilie Le Maout ◽  
Simon Lo Vecchio ◽  
Praveen Kumar Korla ◽  
Jim Jinn-Chyuan Sheu ◽  
Daniel Riveline
Keyword(s):  
Cell Orientation ◽  
Flat Surfaces ◽  
Chemical Gradients ◽  
Biological Phenomena ◽  
Squamous Cancer ◽  
Direct Cell ◽  
Cell Motion ◽  
Cancerous Cells

AbstractCell motility is essential in a variety of biological phenomena ranging from early development to organ homeostasis and diseases. This phenomenon was so far mainly studied and characterized on flat surfaces in vitro whereas this situation is rarely seen in vivo. Recently, cell motion in 3D microfabricated channels was reported to be possible, and it was shown that confined cells push on walls. However, rules setting cell directions in this context were not characterized yet. Here, we show by using assays that ratchetaxis operates in 3D ratchets on fibroblasts and on epithelial cancerous cells. Open ratchets rectify cell motion, whereas closed ratchets impose a direct cell migration along channels set by the cell orientation at the channel entry point. We also show that nuclei are pressed at constrictions zones through mechanisms involving dynamic asymmetries of focal contacts, stress fibers, and intermediate filaments. Interestingly, cells do not pass these constricting zones when defective in the keratin fusion implicated in squamous cancer. By combining ratchetaxis with chemical gradients, we finally report that cells are sensitive to local asymmetries in confinement and that topological and chemical cues may be encoded differently by cells. Altogether our ratchet channels could mimic small blood vessels where cells are confined: cells would probe local asymmetries which would determine their entry into tissues and direction. Our results could shed light on invasions mechanisms in cancer.

scholarly journals Reversible programming of pluripotent cell differentiation

2000 ◽  
Vol 113 (3) ◽  
pp. 555-566 ◽  
Author(s):  
J. Lake ◽  
J. Rathjen ◽  
J. Remiszewski ◽  
P.D. Rathjen
Keyword(s):  
Gene Expression ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Primitive Endoderm ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Differentiation Potential

We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors. EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.

Tests by Tissue Culture Methods on the Nature of Immunity Transplanted Skin

1948 ◽  
Vol s3-89 (7) ◽  
pp. 239-252
Author(s):  
P. B. MEDAWAR
Keyword(s):  
Tissue Culture ◽  
Cell Division ◽  
Epidermal Cell ◽  
Cell Suspensions ◽  
Acquired Immunity ◽  
Culture Methods ◽  
Division Frequency

The transplantation of skin from one rabbit to another elicits a reaction that conforms in main outline with that of an actively acquired immunity. The experiments described in this paper were designed to test the hypothesis that the regression of such grafts is secured by the action of antibodies demonstrable in vitro. Skin from adult rabbits has therefore been cultivated in the presence of serum and growing mesenchymal tissues derived solely from rabbits heavily and specifically immunized against it. Immune sera and tissues are without effect on the survival, cell-division frequency and migratory activities of explanted skin, and agglutinins for epidermal cell suspensions are not demonstrable in immune sera. With certain stated qualifications, it has therefore been concluded that the occurrence of free antibodies is not a sufficient explanation of the regression of skin homografts in vivo.

Tough Double-Network Hydrogels as Scaffolds for Tissue Engineering

2012 ◽  
pp. 213-222
Author(s):  
Jing Jing Yang ◽  
Jian Fang Liu ◽  
Takayuki Kurokawa ◽  
Nobuto Kitamura ◽  
Kazunori Yasuda ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Three Dimensional ◽  
Cartilage Regeneration ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Living Tissue ◽  
Double Network ◽  
Dimensional Network

Hydrogels are used as scaffolds for tissue engineering in vitro & in vivo because their three-dimensional network structure and viscoelasticity are similar to those of the macromolecular-based extracellular matrix (ECM) in living tissue. Especially, the synthetic hydrogels with controllable and reproducible properties were used as scaffolds to study the behaviors of cells in vitro and implanted test in vivo. In this review, two different structurally designed hydrogels, single-network (SN) hydrogels and double-network (DN) hydrogels, were used as scaffolds. The behavior of two cell types, anchorage-dependent cells and anchorage-independent cells, and the differentiation behaviors of embryoid bodies (EBs) were investigated on these hydrogels. Furthermore, the behavior of chondrocytes on DN hydrogels in vitro and the spontaneous cartilage regeneration induced by DN hydrogels in vivo was examined.

Refining In Vitro Neurotoxicity Testing — The Development of Blood–Brain Barrier Models

2003 ◽  
Vol 31 (3) ◽  
pp. 273-276 ◽  
Author(s):  
Hanna Tähti ◽  
Heidi Nevala ◽  
Tarja Toimela
Keyword(s):  
Blood Brain Barrier ◽  
Cell Lines ◽  
Three Dimensional ◽  
Brain Barrier ◽  
Culture Methods ◽  
Blood Brain ◽  
Capillary Endothelial Cells ◽  
In Vitro Bbb Model

The purpose of this paper is to review the current state of development of advanced in vitro blood–brain barrier (BBB) models. The BBB is a special capillary bed that separates the blood from the central nervous system (CNS) parenchyma. Astrocytes maintain the integrity of the BBB, and, without astrocytic contacts, isolated brain capillary endothelial cells in culture lose their barrier characteristics. Therefore, when developing in vitro BBB models, it is important to add astrocytic factors into the culture system. Recently, novel filter techniques and co-culture methods have made it possible to develop models which resemble the in vivo functions of the BBB in an effective way. With a BBB model, kinetic factors can be added into the in vitro batteries used for evaluating the neurotoxic potential of chemicals. The in vitro BBB model also represents a useful tool for the in vitro prediction of the BBB permeability of drugs, and offers the possibility to scan a large number of drugs for their potential to enter the CNS. Cultured monolayers of brain endothelial cell lines or selected epithelial cell lines, combined with astrocyte and neuron cultures, form a novel three-dimensional technique for the screening of neurotoxic compounds.

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2740-2749 ◽  
Author(s):  
CD Helgason ◽  
G Sauvageau ◽  
HJ Lawrence ◽  
C Largman ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Embryoid Bodies ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

scholarly journals Generation and miRNA Characterization of Equine Induced Pluripotent Stem Cells Derived from Fetal and Adult Multipotent Tissues

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Laís Vicari de Figueiredo Pessôa ◽  
Pedro Ratto Lisboa Pires ◽  
Maite del Collado ◽  
Naira Caroline Godoy Pieri ◽  
Kaiana Recchia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Mirna Profile ◽  
Patient Specific ◽  
Induced Pluripotent

Introduction. Pluripotent stem cells are believed to have greater clinical potential than mesenchymal stem cells due to their ability to differentiate into almost any cell type of an organism, and since 2006, the generation of patient-specific induced pluripotent stem cells (iPSCs) has become possible in multiple species. Objectives. We hypothesize that different cell types respond differently to the reprogramming process; thus, the goals of this study were to isolate and characterize equine adult and fetal cells and induce these cells to pluripotency for future regenerative and translational purposes. Methods. Adult equine fibroblasts (eFibros) and mesenchymal cells derived from the bone marrow (eBMmsc), adipose tissue (eADmsc), and umbilical cord tissue (eUCmsc) were isolated, their multipotency was characterized, and the cells were induced in vitro into pluripotency (eiPSCs). eiPSCs were generated through a lentiviral system using the factors OCT4, SOX2, c-MYC, and KLF4. The morphology and in vitro pluripotency maintenance potential (alkaline phosphatase detection, embryoid body formation, in vitro spontaneous differentiation, and expression of pluripotency markers) of the eiPSCs were characterized. Additionally, a miRNA profile analysis of the mesenchymal and eiPSCs was performed. Results. Multipotent cells were successfully isolated, but the eBMmsc failed to generate eiPSCs. The eADmsc-, eUCmsc-, and eFibros-derived iPSCs were positive for alkaline phosphatase, OCT4 and NANOG, were exclusively dependent on bFGF, and formed embryoid bodies. The miRNA profile revealed a segregated pattern between the eiPSCs and multipotent controls: the levels of miR-302/367 and the miR-92 family were increased in the eiPSCs, while the levels of miR-23, miR-27, and miR-30, as well as the let-7 family were increased in the nonpluripotent cells. Conclusions. We were able to generate bFGF-dependent iPSCs from eADmsc, eUCmsc, and eFibros with human OSKM, and the miRNA profile revealed that clonal lines may respond differently to the reprogramming process.

324 CELL LINES DERIVED FROM MAMMALIAN PARTHENOGENETIC EMBRYOS DISPLAY ABNORMAL CHROMOSOME COMPLEMENTS AND ABERRANT CENTRIOLE NUMBER

2010 ◽  
Vol 22 (1) ◽  
pp. 318
Author(s):  
T. A. L. Brevini ◽  
G. Pennarossa ◽  
A. Vanelli ◽  
G. Tettamanti ◽  
L. Bogliolo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Embryoid Body ◽  
Mammalian Species ◽  
High Rate ◽  
Primary Fibroblast ◽  
Thin Sections ◽  
Paternal Contribution ◽  
Parthenogenetic Embryos

Mature oocytes can be activated in vitro, leading to the generation of parthenotes that will develop in culture forming blastocysts morphologically indistinguishable from those derived from fertilized eggs. Parthenotes have been used as a source of pluripotent cells that show the traditional features associated with their biparental counterpart: expression of totipotency markers, telomerase activity, embryoid body formation, in vitro differentiation and, in most cases, teratoma formation. However, many aspects still need to be elucidated and, in particular, little attention has been paid to the inci- dence of aneuploidy in these cells. Limited data available for parthenotes derived from different mammalian species indicate a high rate of aneuploidy, whichis consideredtobecaused by the lackofthe paternal contribution, because alterations of the centrosome are knowntolead to multipolar spindles that, in turn, cause aneuploid cells. In this study, we analyzed the rate of aneuploidy and centriole distribution (as a marker of centrosome anomalies) in pluripotent cell lines (pSC) previously derived in our laboratory from pig parthenogenetic embryos and in primary fibroblast cultures and sections obtained from sheep parthenogenetic fetuses (n = 3) that reached 24 days of development in vivo. This protocol was chosen to separate the effect related tooocyte activation from those of the procedures used to derive pSC lines. Centriole number and distribution were assessed both by immunocy- tochemical analysis using an anti-centrin-1 antibody (1 : 200, Abcam, Cambridge, UK) and an appropriate secondary antibody, and by ultrastructural evaluation of thin sections, using a Jeol 1010 EX electron microscope (Jeol, Tokyo, Japan). Karyotyping was performed on mitotically active cells. Metaphases were fully karyotyped under a Leica HC microscope (Wetzlar, Germany). Images were then captured with a Leica DC250 digital camera and cells karyotyped using the Leica CW4000 Karyo software. The results obtained indicate that cell lines of parthenogenetic origin have, in all examined cases, an incidence of aneuploidy significantly higher than that of their respective controls. In particular, although the diploid configuration represented the modal value, the majority of the cells displayed a consistently lower number of chromosomes, between <1N (hypohaploid) and >1N to <2N (hypodiploid).This resultis possibly related toa lossofchromosomes during the mitotic process.Ahigher incidence ofmultiple centrioles was also detected, suggesting that aneuploidy may be related to the lack of paternal contribution that results in abnormal centrosome formation, incorrect control of the process of spindle rearrangement, and consequent chromosomal malsegregation.Abnormal segregation and multicentriolar distribution were not limited to parthenogenetic cell lines but was observed in parthenotes as well, indicating that culture artifacts are unlikely to be the cause. PUR 2007, PUR 2008.

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