scholarly journals Direct repression of Nanog and Oct4 by OTX2 modulates contribution of epiblast-derived cells to germline and somatic lineage

Development ◽  
2021 ◽  
Author(s):  
Luca Giovanni Di Giovannantonio ◽  
Dario Acampora ◽  
Daniela Omodei ◽  
Vincenzo Nigro ◽  
Pasquale Barba ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Regulatory Network ◽  
Primordial Germ Cells ◽  
Culture Conditions ◽  
Specific Cell ◽  
Temporal Window ◽  
Pluripotency Gene ◽  
Gene Regulatory ◽  
Cell Culture Conditions

In mammals the pre-gastrula proximal epiblast gives rise to Primordial Germ Cells (PGCs) or somatic precursors in response to BMP4 and WNT signaling. Entry into the germline requires activation of a naïve-like pluripotency gene regulatory network (GRN). Recent work showed that suppression of OTX2 expression in the epiblast by BMP4 allows cells to develop a PGC fate in a precise temporal window. However, the mechanisms by which OTX2 suppresses PGC fate are unknown. Here we show that OTX2 prevents epiblast cells from activating the pluripotency GRN by direct repression of Oct4 and Nanog. Loss of this control during PGC differentiation in vitro causes widespread activation of the pluripotency GRN and deregulated response to LIF, BMP4 and WNT signaling. These abnormalities, in specific cell culture conditions, result in massive germline entry at the expense of somatic mesoderm differentiation. Increased generation of PGCs occurs also in mutant embryos. We propose that the OTX2 repressive control of Oct4 and Nanog is at the basis of the mechanism determining epiblast contribution to germline and somatic lineage.

Electron microscopic studies on the structure of motile primordial germ cells of Xenopus laevis in vitro

Development ◽  
1978 ◽  
Vol 46 (1) ◽  
pp. 119-133
Author(s):  
Janet Heasman ◽  
C. C. Wylie
Keyword(s):  
Xenopus Laevis ◽  
Germ Cells ◽  
Primordial Germ Cells ◽  
Structural Features ◽  
Culture Conditions ◽  
Structural Basis ◽  
Electron Microscopic ◽  
Microscopic Studies

Primordial germ cells (PGCs) of Xenopus laevis have been isolated from early embryos and kept alive in vitro, in order to study the structural basis of their motility, using the transmission and scanning electron microscope. The culture conditions used mimicked as closely as possible the in vivo environment of migrating PGCs, in that isolated PGCs were seeded onto monolayers of amphibian mesentery cells. In these conditions we have demonstrated that: (a) No significant differences were found between the morphology of PGCs in vitro and in vivo. (b) Structural features involved in PGC movement in vitro include (i) the presence of a filamentous substructure, (ii) filopodial and blunt cell processes, (iii) cell surface specializations. These features are also characteristic of migratory PGCs studied in vivo. (c) PGCs in vitro have powers of invasion similar to those of migrating PGCs in vivo. They occasionally become completely surrounded by cells of the monolayer and, in this situation, bear striking resemblance to PGCs moving between mesentery cells to the site of the developing gonad in stage-44 tadpoles. We conclude that as far as it is possible to assess, the behaviour of isolated PGCs in these in vitro conditions mimics their activities in vivo. This allows us to study the ultrastructural basis of their migration.

scholarly journals Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 934 ◽  
Author(s):  
Ecke ◽  
Lutter ◽  
Scholka ◽  
Hansch ◽  
Becker ◽  
...  
Keyword(s):  
Articular Chondrocytes ◽  
Culture Conditions ◽  
Serum Supplement ◽  
3 Dimensional ◽  
The Matrix ◽  
Differentiation Degree ◽  
Overlay Technique ◽  
Fetal Bovine ◽  
Cell Culture Conditions

Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement.

scholarly journals Ground rules of the pluripotency gene regulatory network

2017 ◽  
Vol 18 (3) ◽  
pp. 180-191 ◽  
Author(s):  
Mo Li ◽  
Juan Carlos Izpisua Belmonte
Keyword(s):  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Pluripotency Gene ◽  
Gene Regulatory

scholarly journals Defining cell culture conditions to improve human norovirus infectivity assays

2013 ◽  
Vol 67 (4) ◽  
pp. 863-868 ◽  
Author(s):  
T. M. Straub ◽  
J. R. Hutchison ◽  
R. A. Bartholomew ◽  
C. O. Valdez ◽  
N. B. Valentine ◽  
...  
Keyword(s):  
Cell Line ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Qrt Pcr ◽  
Infectivity Assay ◽  
Polymerase Chain ◽  
Single Laboratory ◽  
Cell Culture Conditions ◽  
Human Intestinal Cells

Significant difficulties remain for determining whether human noroviruses (hNoV) recovered from water, food, and environmental samples are infectious. Three-dimensional (3-D) tissue culture of human intestinal cells has shown promise in developing an infectivity assay, but reproducibility, even within a single laboratory, remains problematic. From the literature and our observations, we hypothesized that the common factors that lead to more reproducible hNoV infectivity in vitro requires that the cell line be (1) of human gastrointestinal origin, (2) expresses apical microvilli, and (3) be a positive secretor cell line. The C2BBe1 cell line, which is a brush-border producing clone of Caco-2, meets these three criteria. When challenged with Genogroup II viruses, we observed a 2 Log10 increase in viral RNA titer. A passage experiment with GII viruses showed evidence of the ability to propagate hNoV by both quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microscopy. In our hands, using 3-D C2BBe1 cells improves reproducibility of the infectivity assay for hNoV, but the assay can still be variable. Two sources of variability include the cells themselves (mixed phenotypes of small and large intestine) and initial titer measurements using qRT-PCR that measures all RNA vs. plaque assays that measure infectious virus.

scholarly journals Generation of Miniaturized Ovaries by in Vitro Culture From Mouse Gonads

2021 ◽  
Author(s):  
Si Won Jang ◽  
Hoon Jang ◽  
Hyun Woo Choi
Keyword(s):  
Ovarian Development ◽  
Primordial Germ Cells ◽  
Nutrient Deficiency ◽  
Culture Conditions ◽  
The Body ◽  
Reproductive Age ◽  
Considerable Research ◽  
Complex Process ◽  
Genetic And Environmental Factors

Abstract The incidence of infertility among individuals of reproductive age has been growing due to genetic and environmental factors, and considerable research efforts are focused on solving this issue. Ovarian development is an overly complex process in the body, involving the interaction between primordial germ cells and gonad somatic cells. However, follicles located in the center of the in vitro ovary are poorly formed or die owing to ovarian complexity, nutrient deficiency, and signaling deficiency. In the present study, we optimized methods for dissociating gonads and culture conditions for the in vitro generation of miniaturized ovaries. The gonads from embryos were dissociated into cell masses and cultured on a Transwell-COL membrane for 3~5 weeks. Approximately 12 follicles were present per in vitro ovary. We observed that miniaturized ovaries successfully matured to MII oocytes in vitro from 150 to 100 µm gonad masses. This method will be useful for investigating follicle development and oocyte production.

scholarly journals Genetic signatures of evolution of the pluripotency gene regulating network across mammals

2020 ◽  
Author(s):  
Yoshinori Endo ◽  
Ken-ichiro Kamei ◽  
Miho Inoue-Murayama
Keyword(s):  
Gene Regulatory Network ◽  
Regulatory Network ◽  
Mammalian Species ◽  
Rapid Evolution ◽  
Network Evolution ◽  
Downstream Targets ◽  
The Core ◽  
Regulatory Circuit ◽  
Pluripotency Gene ◽  
Gene Regulatory

AbstractMammalian pluripotent stem cells (PSCs) have distinct molecular and biological characteristics, but we lack a comprehensive understanding of regulatory network evolution in mammals. Here, we applied a comparative genetic analysis of 134 genes constituting the pluripotency gene regulatory network across 48 mammalian species covering all the major taxonomic groups. We found evolutionary conservation in JAK-STAT and PI3K-Akt signaling pathways, suggesting equivalent capabilities in self-renewal and proliferation of mammalian PSCs. On the other hand, we discovered rapid evolution of the downstream targets of the core regulatory circuit, providing insights into variations of characteristics. Our data indicate that the variations in the PSCs characteristics may be due to positive selections in the downstream targets of the core regulatory circuit. We further reveal that positively selected genes can be associated with species unique adaptation that is not dedicated to regulation of PSCs. These results provide important insight into the evolution of the pluripotency gene regulatory network underlying variations in characteristics of mammalian PSCs.

scholarly journals The G-box transcriptional regulatory code in Arabidopsis

2017 ◽  
Author(s):  
Daphne Ezer ◽  
Samuel JK Shepherd ◽  
Anna Brestovitsky ◽  
Patrick Dickinson ◽  
Sandra Cortijo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Network ◽  
Environmental Stressors ◽  
Sequence Motifs ◽  
Transcription Pattern ◽  
Expression Of Genes ◽  
Transcriptional Regulatory ◽  
Flanking Sequences ◽  
Gene Regulatory

ABSTRACTPlants have significantly more transcription factor (TF) families than animals and fungi, and plant TF families tend to contain more genes—these expansions are linked to adaptation to environmental stressors (1, 2). Many TF family members bind to similar or identical sequence motifs, such as G-boxes (CACGTG), so it is difficult to predict regulatory relationships. We determine that the flanking sequences near G-boxes help determine in vitro specificity, but that this is insufficient to predict the transcription pattern of genes near G-boxes. Therefore, we construct a gene regulatory network that identifies the set of bZIPs and bHLHs that are most predictive of the gene expression of genes downstream of perfect G-boxes. This network accurately predicts transcriptional patterns and reconstructs known regulatory subnetworks. Finally, we present Ara-BOX-cis (araboxcis.org), a website that provides interactive visualisations of the G-box regulatory network, a useful resource for generating predictions for gene regulatory relations.

Laser microstructured scaffolds for tissue engineering under dynamic cell culture conditions

2020 ◽  
Author(s):  
Ελευθερία Μπαμπαλιάρη
Keyword(s):  
Tissue Engineering ◽  
Cell Culture ◽  
Culture Conditions ◽  
Dynamic Cell ◽  
Cell Culture Conditions

Παρόλο που το περιφερικό νευρικό σύστημα εμφανίζει υψηλότερο ρυθμό αναγέννησης από εκείνο του κεντρικού νευρικού συστήματος μέσω αυθόρμητης αναγέννησης μετά από έναν τραυματισμό, η καθοδηγούμενη αξονική νευρική αναγέννηση και η λειτουργική αποκατάσταση είναι αρκετά σπάνια. Συνεπώς, η ανάπτυξη επιτυχημένων μεθόδων για την καθοδήγηση της νευρικής ανάπτυξης, «in vitro», είναι υψίστης σημασίας. Έχει αναφερθεί λεπτομερώς ότι η τοπογραφία του υποστρώματος επηρεάζει την ανάπτυξη, τον προσανατολισμό και τη διαφοροποίηση των νευρικών κυττάρων. Ωστόσο, η συνδυασμένη δράση της διατμητικής τάσης και της τοπογραφίας του υποστρώματος στην νευρική ανάπτυξη έχει ελάχιστα μελετηθεί, παρόλο που οι διατμητικές τάσεις είναι ευρέως γνωστό ότι διαδραματίζουν καθοριστικό ρόλο στην οργάνωση, ανάπτυξη και λειτουργία των ιστών. Σε αυτή τη μελέτη, ένα σύστημα μικροροών ακριβούς ελεγχόμενης ροής με συγκεκριμένους ειδικά σχεδιασμένους θαλάμους, που ενσωματώνουν μικροδομημένα υποστρώματα λέιζερ, αναπτύχθηκε για να μελετηθεί η συνδυασμένη δράση της διατμητικής τάσης και της τοπογραφίας υποστρώματος στην ανάπτυξη, στον προσανατολισμό, στην επιμήκυνση και στη διαφοροποίηση νευρικών κυττάρων. Πολυμερικά μικροδομημένα υποστρώματα, με ελεγχόμενη γεωμετρία και κανονικότητα μοτίβου, κατασκευάστηκαν με χρήση υπερβραχέων παλμών λέιζερ. Πραγματοποιήθηκε συγκριτική μελέτη μεταξύ στατικών και δυναμικών κυτταρικών καλλιεργειών για να αξιολογηθεί η συνεργατική ή ανταγωνιστική επίδραση της διατμητικής τάσης και της τοπογραφίας στη συμπεριφορά των νευρικών κυττάρων. Τα αποτελέσματα της κυτταρικής καλλιέργειας συμπληρώθηκαν με υπολογιστικές προσομοιώσεις ροής με σκοπό τον ακριβή υπολογισμό των αντίστοιχων τιμών διατμητικής τάσης.

scholarly journals Enhancement Of Dermal Fibroblast Isolation Method

2015 ◽  
Vol 16 (1) ◽  
pp. 65-69
Author(s):  
Milan Zaric ◽  
Ivana Nikolic ◽  
Ivanka Zelen ◽  
Marina Mitrovic ◽  
Zoran Milosavljevic
Keyword(s):  
Dermal Fibroblast ◽  
Petri Dish ◽  
Culture Conditions ◽  
Dermal Fibroblasts ◽  
Enzyme Digestion ◽  
Digestion Method ◽  
Primary Isolation ◽  
Donor Cells ◽  
Cell Culture Conditions

ABSTRACTCultivated fibroblasts have been widely used in a large number of in vitro studies. Although they readily proliferate under cell culture conditions, improvements in methods for their isolation are necessary. Here, we present our modified enzyme digestion method and compare its efficiency with commonly used techniques.Three foreskin samples from young, middle-aged and old donors were used. The classical explant, standard enzyme digestion method with collagenase and our improved enzyme digestion method were compared for efficiency of fibroblast isolation and the time needed to achieve 95% confluence in a 30-mm Petri dish.The explant method was the slowest to achieve fibroblast confluence, especially with the tissues from the older donors (up to 23 days). With the standard enzyme digestion method, the skin tissue was partially digested, but the fibroblasts reached confluence much faster (the younger donor cells needed approximately 7 days to reach confluence). Our modified “mixed” enzyme digestion method was the fastest (the fibroblasts from the younger donors needed up to 5 days to reach confluence).For studies requiring the primary isolation and cultivation of dermal fibroblasts, the best method to achieve this goal is the tissue digestion method with the multiple enzyme solution.

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