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 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.

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.

Designing and Engineering Stem Cell Niches

MRS Bulletin ◽  
2010 ◽  
Vol 35 (8) ◽  
pp. 591-596 ◽  
Author(s):  
Ana I. Teixeira ◽  
Ola Hermanson ◽  
Carsten Werner
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Chemical Engineering ◽  
Polyglycolic Acid ◽  
Differentiated Cells ◽  
Extracellular Matrix Components ◽  
Stem Cell Niches

AbstractStem cells have received a lot of attention due to great promises in medical treatment, for example, by replacing lost and sick cells and re-constituting cell populations. There are several classes of stem cells, including embryonic, fetal, and adult tissue specific. More recently, the generation of so-called induced pluripotent stem (iPS) cells from differentiated cells has been established. Common criteria for all types of stem cells include their ability to self-renew and to retain their ability to differentiate in response to specific cues. These characteristics, as well as the instructive steering of the cells into differentiation, are largely dependent on the microenvironment surrounding the cells. Such “stem cell friendly” microenvironments, provided by structural and biochemical components, are often referred to as niches. Biomaterials offer attractive solutions to engineer functional stem cell niches and to steer stem cell state and fatein vitroas well asin vivo. Among materials used so far, promising results have been achieved with low-toxicity and biodegradable polymers, such as polyglycolic acid and related materials, as well as other polymers used as structural “scaffolds” for engineering of extracellular matrix components. To improve the efficiency of stem cell control and the design of the biomaterials, interfaces among stem cell research, developmental biology, regenerative medicine, chemical engineering, and materials research are rapidly developing. Here we provide an introduction to stem cell biology and principles of niche engineering and give an overview of recent advancements in stem cell niche engineering from two stem cell systems—blood and brain.

scholarly journals Platelet-Derived Growth Factor Receptor Alpha as a Marker of Mesenchymal Stem Cells in Development and Stem Cell Biology

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Ramin M. Farahani ◽  
Munira Xaymardan
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Growth Factor Receptor ◽  
Stem Cell Marker ◽  
Factor Receptor Alpha ◽  
Lines Of Evidence

Three decades on, the mesenchymal stem cells (MSCs) have been intensively researched on the bench top and used clinically. However, ambiguity still exists in regard to their anatomical locations, identities, functions, and extent of their differentiative abilities. One of the major impediments in the quest of the MSC research has been lack of appropriatein vivomarkers. In recent years, this obstacle has been resolved to some degree as PDGFRαemerges as an important mesenchymal stem cell marker. Accumulating lines of evidence are showing that the PDGFRα+cells reside in the perivascular locations of many adult interstitium and fulfil the classic concepts of MSCsin vitroandin vivo. PDGFRαhas long been recognised for its roles in the mesoderm formation and connective tissue development during the embryogenesis. Current review describes the lines of evidence regarding the role of PDGFRαin morphogenesis and differentiation and its implications for MSC biology.

scholarly journals Gastric Cancer Stem Cells

2008 ◽  
Vol 26 (17) ◽  
pp. 2876-2882 ◽  
Author(s):  
Shigeo Takaishi ◽  
Tomoyuki Okumura ◽  
Timothy C. Wang
Keyword(s):  
Gastric Cancer ◽  
Stem Cells ◽  
Stem Cell ◽  
Cancer Stem Cells ◽  
Cancer Stem Cell ◽  
Cell Biology ◽  
Cancer Biology ◽  
Human Cancer ◽  
Immunodeficient Mice

Cancer stem cells are defined as the unique subpopulation in the tumors that possess the ability to initiate tumor growth and sustain self-renewal as well as metastatic potential. Accumulating evidence in recent years strongly indicate the existence of cancer stem cells in solid tumors of a wide variety of organs. In this review, we will discuss the possible existence of a gastric cancer stem cell. Our recent data suggest that a subpopulation with a defined marker shows spheroid colony formation in serum-free media in vitro, as well as tumorigenic ability in immunodeficient mice in vivo. We will also discuss the possible origins of the gastric cancer stem cell from an organ-specific stem cell versus a recently recognized new candidate bone marrow–derived cell (BMDC). We have previously shown that BMDC contributed to malignant epithelial cells in the mouse model of Helicobacter-associated gastric cancer. On the basis of these findings from animal model, we propose that a similar phenomenon may also occur in human cancer biology, particularly in the cancer origin of other inflammation-associated cancers. The expanding research field of cancer stem-cell biology may offer a novel clinical apparatus to the diagnosis and treatment of cancer.

scholarly journals Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells

2016 ◽  
Author(s):  
Yu-Ting L. Dingle ◽  
Katherine B. Xiong ◽  
Jason T. Machan ◽  
Kimberly A. Seymour ◽  
Debra Ellisor ◽  
...  
Keyword(s):  
Stem Cell ◽  
Sonic Hedgehog ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Systematic Random Sampling ◽  
Brain Science ◽  
Fibroblast Growth Factor 8 ◽  
Brown University

AbstractDopamine (DA) neuron subtypes modulate specific physiological functions and are involved in distinct neurological disorders. Embryonic stem cell (ESC) derived DA neurons have the potential to aid in the study of disease mechanisms, drug discovery, and possibly cell replacement therapies. DA neurons can be generated from ESCs in vitro, but the subtypes of ESC-derived DA neurons have not been investigated in detail despite the diversity of DA neurons observed in vivo. Due to cell culture heterogeneity, sampling methods applied to ESC-derived cultures can be ambiguous and potentially biased. Therefore, we developed a quantification method to capture the depth of DA neuron production in vitro by estimating the error associated with systematic random sampling. Using this method, we quantified calbindin+ and calretinin+ subtypes of DA neurons generated from mouse ESCs. We found a higher production of the calbindin+ subtype (11−27%) compared to the calretinin+ subtype (2-13%) of DA neuron; in addition, DA neurons expressing neither subtype marker were also generated. We then examined whether exogenous sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) affected subtype generation. Our results demonstrate that exogenous SHH and FGF8 did not alter DA neuron subtype generation in vitro. These findings suggest that a deeper understanding DA neuron derivation inclusive of mechanisms that govern the in vitro subtype specification of ESC-derived DA neurons is required.NoteAll research was planned and conducted while members were at Brown UniversityResearch fundingNIH/NCRR/NIGMS RI Hospital COBRE Center for Stem Cell Biology (8P20GM103468-04) (MZ) Brown Institute for Brain Science Pilot Grant (4-63662) (MZ/DHK)

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.

scholarly journals Message in a Bottle: Upgrading Cardiac Repair into Rejuvenation

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 724 ◽  
Author(s):  
Carolina Balbi ◽  
Ambra Costa ◽  
Lucio Barile ◽  
Sveva Bollini
Keyword(s):  
Stem Cell ◽  
Cell Biology ◽  
Cardiac Tissue ◽  
Optimal Choice ◽  
Cardiac Repair ◽  
Cardiomyocyte Proliferation ◽  
Therapeutic Approaches ◽  
Comprehensive Overview ◽  
Future Therapy

Ischaemic cardiac disease is associated with a loss of cardiomyocytes and an intrinsic lack of myocardial renewal. Recent work has shown that the heart retains limited cardiomyocyte proliferation, which remains inefficient when facing pathological conditions. While broadly active in the neonatal mammalian heart, this mechanism becomes quiescent soon after birth, suggesting loss of regenerative potential with maturation into adulthood. A key question is whether this temporary regenerative window can be enhanced via appropriate stimulation and further extended. Recently the search for novel therapeutic approaches for heart disease has centred on stem cell biology. The “paracrine effect” has been proposed as a promising strategy to boost endogenous reparative and regenerative mechanisms from within the cardiac tissue by exploiting the modulatory potential of soluble stem cell-secreted factors. As such, growing interest has been specifically addressed towards stem/progenitor cell-secreted extracellular vesicles (EVs), which can be easily isolated in vitro from cell-conditioned medium. This review will provide a comprehensive overview of the current paradigm on cardiac repair and regeneration, with a specific focus on the role and mechanism(s) of paracrine action of EVs from cardiac stromal progenitors as compared to exogenous stem cells in order to discuss the optimal choice for future therapy. In addition, the challenges to overcoming translational EV biology from bench to bedside for future cardiac regenerative medicine will be discussed.

The Emerging Role of Stem Cells in Regenerative Dentistry

2020 ◽  
Vol 20 (4) ◽  
pp. 259-268 ◽  
Author(s):  
Paolo Capparè ◽  
Giulia Tetè ◽  
Maria Teresa Sberna ◽  
Paola Panina-Bordignon
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Specific Cell ◽  
Cell Plasticity ◽  
Regenerative Dentistry ◽  
Cell Sources ◽  
Induced Pluripotent

Progress of modern dentistry is accelerating at a spectacular speed in the scientific, technological and clinical areas. Practical examples are the advancement in the digital field, which has guaranteed an average level of prosthetic practices for all patients, as well as other scientific developments, including research on stem cell biology. Given their plasticity, defined as the ability to differentiate into specific cell lineages with a capacity of almost unlimited self-renewal and release of trophic/immunomodulatory factors, stem cells have gained significant scientific and commercial interest in the last 15 years. Stem cells that can be isolated from various tissues of the oral cavity have emerged as attractive sources for bone and dental regeneration, mainly due to their ease of accessibility. This review will present the current understanding of emerging conceptual and technological issues of the use of stem cells to treat bone and dental loss defects. In particular, we will focus on the clinical application of stem cells, either directly isolated from oral sources or in vitro reprogrammed from somatic cells (induced pluripotent stem cells). Research aimed at further unraveling stem cell plasticity will allow to identify optimal stem cell sources and characteristics, to develop novel regenerative tools in dentistry.

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