scholarly journals Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice

F1000Research ◽  
2019 ◽  
Vol 8 ◽  
pp. 1983
Author(s):  
Christopher Blackwood
Keyword(s):  
Stem Cell ◽  
Neural Stem Cell ◽  
Cell Biology ◽  
Stem Cell Biology ◽  
Ganglionic Eminence ◽  
Lateral Ganglionic Eminence ◽  
Epidermal Growth ◽  
Dissection Technique

Background: The neurosphere assay is a powerful tool to study neural stem cell biology. The objective of this protocol is to create a simple and rapid approach to generate neurospheres from the dorsal lateral ganglionic eminence of late embryonic (day 17) mice. This method predicts the average number of neurospheres and provides an approximation of its expected size after 7 days in vitro. Characterization of numbers and sizes will provide investigators with quantitative data to advise on the implementation of downstream applications, including immnocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro. Furthermore, we found this method yields different sizes of neurospheres. Lastly, using an anti-GFAP antibody, we confirm that these neurospheres can be used for immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of neurospheres that will be useful for further advances in the area of stem cell biology.

scholarly journals Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice

F1000Research ◽  
2020 ◽  
Vol 8 ◽  
pp. 1983
Author(s):  
Christopher Blackwood
Keyword(s):  
Stem Cell ◽  
Lateral Ventricle ◽  
Cell Biology ◽  
Lateral Ganglionic Eminence ◽  
Stepwise Method ◽  
Specialized Equipment ◽  
Epidermal Growth ◽  
Dissection Technique ◽  
Neurosphere Assay

Background: The neurosphere assay is a powerful in vitro tool to investigate neural stem cells in the dorsal lateral ventricle (dLGE). In the dLGE, metrics of sizes and numbers of neurospheres generated using this assay has not been completely characterized. The objective of this protocol is to provide a stepwise method from a single isolation that predicts the average number of neurospheres generated and to estimate an approximation of its sizes after several days in vitro. The advantage of this protocol is that no expensive and specialized equipment is needed for tissue isolation. Estimates about the numbers and sizes of neurospheres will provide investigators with quantitative data to advise on how much starting dLGE tissue is required to generate the appropriate number of spheres for the implementation of downstream applications, including immunocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro (DIV). Furthermore, we observed that this method yields an average range of neurospheres sizes greater than 50 μm, but less than 100 μm after 7 DIV. Lastly, using an anti-GFAP antibody, we show that these neurospheres can be stained, confirming their use in future immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of primary neurospheres that will be useful for further advances in the area of stem cell biology.

scholarly journals Role of nuclear receptors in neural stem cell biology of the mammalian central nervous system

2016 ◽  
Author(s):  
Αθανάσιος Στεργιόπουλος
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Stem Cell ◽  
Neural Stem Cell ◽  
Nuclear Receptors ◽  
Cell Biology ◽  
Stem Cell Biology ◽  
Mammalian Central Nervous System

Το δυναμικό και η ικανότητα αυτο-ανανέωσης και διαφοροποίησης των νευρικών βλαστικών κυττάρων (ΝΒΚ) ελέγχονται από τη δράση διαφόρων μεταγραφικών παραγόντων και πυρηνικών υποδοχέων, επηρεάζοντας μ ’αυτόν τον τρόπο την ανάπτυξη και τη λειτουργία του κεντρικού νευρικού συστήματος (ΚΝΣ). Στην παρούσα μελέτη χαρακτηρίσαμε τον ορφανό πυρηνικό υποδοχέα NR5A2 (LRH1), ως ένα νέο μόριο το οποίο κατέχει κεντρικό αναπτυξιακό ρόλο στο ΚΝΣ. Με πειράματα υπερ-έκφρασης και αποσιώπησης γονιδίων σε πρωτογενή ΝΒΚ καθώς και με ανάλυση εμβρύων ποντικών στα οποία έχει επιτραπεί η ιστο-ειδική και χρονική εξάλειψη του NR5A2, δείξαμε πως ο NR5A2 είναι ικανός να διακόπτει τον πολλαπλασιασμό των ΝΒΚ, οδηγώντας τα προς τη νευρωνική διαφοροποίηση με την παράλληλη απώλεια των αστροκυττάρων. Σε μηχανιστική βάση, ο NR5A2 ελέγχει αυτούς τους φαινοτύπους μέσω της άμεσης επίδρασής του στον γενετικό τόπο του Ink4/Arf, στο Prox1, το οποίο αποτελεί καθοδικό στόχο των προ-νευρικών γονιδίων, καθώς επίσης και στα σηματοδοτικά μονοπάτια του Notch1 και του JAK/STAT. Αντιθέτως, ο NR5A2 ρυθμίζεται ανοδικά από προ-νευρικά γονίδια και από τα Notch1 και JAK/STAT μονοπάτια. Συμπερασματικά, οι παρατηρήσεις μας προτείνουν τον NR5A2 σαν ένα νέο υποδοχέα-ρυθμιστή της ανάπτυξης του ΚΝΣ, και, σε συνδυασμό με την ανακάλυψη αγωνιστών/ανταγωνιστών του, τον καθιστούν υποψήφιο στόχο στην ανάπτυξη θεραπευτικών στρατηγικών αναγεννητικής ιατρικής του ΚΝΣ.

The Role of Nucleophosmin In Hematopoietic Stem Cells and the Pathogenesis of Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 95-95 ◽  
Author(s):  
Keisuke Ito ◽  
Paolo Sportoletti ◽  
John G Clohessy ◽  
Grisendi Silvia ◽  
Pier Paolo Pandolfi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Cell Biology ◽  
De Novo ◽  
Stem Cell Biology ◽  
Hematopoietic Stem

Abstract Abstract 95 Myelodysplastic syndrome (MDS) is an incurable stem cell disorder characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Nucleophosmin (NPM) is directly implicated in primitive hematopoiesis, the pathogenesis of hematopoietic malignancies and more recently of MDS. However, little is known regarding the molecular role and function of NPM in MDS pathogenesis and in stem cell biology. Here we present data demonstrating that NPM plays a critical role in the maintenance of hematopoietic stem cells (HSCs) and the transformation of MDS into leukemia. NPM is located on chromosome 5q and is frequently lost in therapy-related and de novo MDS. We have previously shown that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment and Npm1+/− mice develop a hematologic syndrome with features of human MDS, including increased susceptibility to leukemogenesis. As HSCs have been demonstrated to be the target of the primary neoplastic event in MDS, a functional analysis of the HSC compartment is essential to understand the molecular mechanisms in MDS pathogenesis. However, the role of NPM in adult hematopoiesis remains largely unknown as Npm1-deficiency leads to embryonic lethality. To investigate NPM function in adult hematopoiesis, we have generated conditional knockout mice of Npm1, using the Cre-loxP system. Analysis of Npm1 conditional mutants crossed with Mx1-Cre transgenic mice reveals that Npm1 plays a crucial role in adult hematopoiesis and ablation of Npm1 in adult HSCs leads to aberrant cycling and followed by apoptosis. Analysis of cell cycle status revealed that HSCs are impaired in their ability to maintain quiescence after Npm1-deletion and are rapidly depleted in vivo as well as in vitro. Competitive reconstitution assay revealed that Npm1 acts cell-autonomously to maintain HSCs. Conditional inactivation of Npm1 leads to an MDS phenotype including a profoundly impaired ability to differentiate into cells of the erythroid lineage, megakaryocyte dyspoiesis and centrosome amplification. Furthermore, Npm1 loss evokes a p53-dependent response and Npm1-deleted HSCs undergo apoptosis in vivo and in vitro. Strikingly, transfer of the Npm1 mutation into a p53-null background rescued the apoptosis of Npm1-ablated HSCs and resulted in accelerated transformation to an aggressive and lethal form of acute myeloid leukemia. Our findings highlight the crucial role of NPM in stem cell biology and identify a new mechanism by which MDS can progress to leukemia. This has important therapeutic implications for de novo MDS as well as therapy-related MDS, which is known to rapidly evolve to leukemia with frequent loss or mutation of TRP53. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reelin function in neural stem cell biology

2002 ◽  
Vol 99 (6) ◽  
pp. 4020-4025 ◽  
Author(s):  
H. M. Kim ◽  
T. Qu ◽  
V. Kriho ◽  
P. Lacor ◽  
N. Smalheiser ◽  
...  
Keyword(s):  
Stem Cell ◽  
Neural Stem Cell ◽  
Cell Biology ◽  
Stem Cell Biology

scholarly journals Advances and Applications in Stem Cell Biology

2012 ◽  
Vol 46 (2) ◽  
pp. 75-80
Author(s):  
Shamoli Bhattacharyya
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Adult Stem Cells ◽  
Great Promise ◽  
Stem Cell Biology ◽  
Self Renewal ◽  
Main Challenge ◽  
Basic Biology

ABSTRACT Mesenchymal stem cells have shown great promise as the source of adult stem cells for regenerative medicine. Present research efforts are directed at isolating these cells from various sources, growing them in vitro and maintaining their pluripotency as well as capacity for self renewal. It is crucial to identify the regulatory molecules which directly or indirectly control the proliferative status or influence the niche microenvironment. The main challenge is to understand the basic biology of the stem cells and manipulate them for further therapeutic applications. Considering their malignant potential, stem cells may be a double edged sword. While the benefits of these cells need to be harnessed judiciously, a significant amount of research is required before embarking on widespread use of this tool for the benefit of humanity. How to cite this article Bhattacharyya S. Advances and Applications in Stem Cell Biology. J Postgrad Med Edu Res 2012;46(2):75-80.

Preclinical Challenges and Clinical Target of Nanomaterials in Regenerative Medicine

2018 ◽  
pp. 1402-1423
Author(s):  
Martin Reinhardt ◽  
Shibashish Giri ◽  
Augustinus Bader
Keyword(s):  
Tissue Engineering ◽  
Stem Cell ◽  
Cell Biology ◽  
Stem Cell Biology ◽  
Organ Engineering ◽  
Cell Therapies ◽  
Existing Problems ◽  
Present Generation

Currently, practical application of nanotechnological approaches and stem cell therapies remains a challenge in both preclinical and clinical settings. Many existing problems in tissue engineering to organ engineering have been solved by the combined approaches of nanotechnology and stem cell biology, but significant barriers remain. Details about the role of various types of nanomaterial in preclinical and clinical research have been reviewed elsewhere, but scant information exists about the influence of nanomaterials on stem cell biology. Herein, the authors highlight the current advances of nanotechnological approaches for expansion, differentiations, harvesting, labeling, imagining, tissue engineering, and organ engineering of different types of stem cells. The preclinical outcome of in vitro and in vivo animal experimentations along with some examples of clinical outcomes of nanomaterials on stem cell research is the main focus of this chapter. This book chapter might be an impetus for the present generation of young scientists to revolutionize the coming generation of effective human healthcare.

Neural Stem Cell Biology

2021 ◽  
pp. 78-85
Author(s):  
Angela Genchi ◽  
Beatrice Von Wunster ◽  
Paola Panina-Bordignon ◽  
Gianvito Martino
Keyword(s):  
Stem Cell ◽  
Neural Stem Cell ◽  
Cell Biology ◽  
Stem Cell Biology
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