scholarly journals Single-Cell Phosphospecific Flow Cytometric Analysis of Canine and Murine Adipose-Derived Stem Cells

2017 ◽  
Vol 2017 ◽  
pp. 1-7 ◽  
Author(s):  
Harumichi Itoh ◽  
Shimpei Nishikawa ◽  
Tomoya Haraguchi ◽  
Yu Arikawa ◽  
Masato Hiyama ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Single Cell ◽  
Stromal Cells ◽  
Flow Cytometric Analysis ◽  
The Other ◽  
Adipose Derived Stem Cells ◽  
Akt Phosphorylation ◽  
Stat Proteins ◽  
Flow Cytometric

This study aimed to demonstrate single-cell phosphospecific flow cytometric analysis of canine and murine adipose-derived stem/stromal cells (ADSCs). ADSCs were obtained from clinically healthy laboratory beagles and C57BL/6 mice. Cell differentiation into adipocytes, osteocytes, and chondrocytes was observed for the cultured canine ADSCs (cADSCs) and murine ADSCs (mADSCs) to determine their multipotency. We also performed single-cell phosphospecific flow cytometric analysis related to cell differentiation and stemness. Cultured cADSCs and mADSCs exhibited the potential to differentiate into adipocytes, osteocytes, and chondrocytes. In addition, single-cell phosphospecific flow cytometric analysis revealed similar β-catenin and Akt phosphorylation between mADSCs and cADSCs. On the other hand, it showed the phosphorylation of different Stat proteins. It was determined that cADSCs and mADSCs show the potential to differentiate into adipocytes, osteocytes, and chondrocytes. Furthermore, a difference in protein phosphorylation between undifferentiated cADSCs and mADSCs was identified.

scholarly journals Secreted factors from dental pulp stem cells improve Sjögren’s syndrome via regulatory T cell-mediated immunosuppression

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mayu Matsumura-Kawashima ◽  
Kenichi Ogata ◽  
Masafumi Moriyama ◽  
Yuka Murakami ◽  
Tatsuya Kawado ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cell Differentiation ◽  
Dental Pulp ◽  
Flow Cytometric Analysis ◽  
The Other ◽  
Activated T Cells ◽  
Expression Levels ◽  
Flow Cytometric ◽  
Secreted Factors

Abstract Background Sjögren’s syndrome (SS) is a chronic autoimmune disease primarily characterized by inflammation in the salivary and lacrimal glands. Activated T cells contribute to disease pathogenesis by producing proinflammatory cytokines, which leads to a positive feedback loop establishment. The study aimed to evaluate the effects of secreted factors derived from dental pulp stem cells (DPSCs) or bone marrow mesenchymal stem cells (BMMSCs) on hyposalivation in SS and to investigate the mechanism involved. Methods Eighty percent confluent stem cells were replenished with serum-free Dulbecco’s modified Eagle’s medium and incubated for 48 h; following which, conditioned media from DPSCs (DPSC-CM) and BMMSCs (BMMSC-CM) were collected. Cytokine array analysis was performed to assess the types of cytokines present in the media. Flow cytometric analysis was performed to evaluate the number of activated T cells cultured in DPSC-CM or BMMSC-CM. Subsequently, DPSC-CM or BMMSC-CM was administered to an SS mouse model. The mice were categorized into the following groups (n = 6 each): non-treatment, Dulbecco’s modified Eagle’s medium (−), BMMSC-CM, and DPSC-CM. Histological analysis of the salivary glands was performed. The gene and protein expression levels of cytokines associated with T helper subsets in the submandibular glands (SMGs) were evaluated. Results DPSC-CM contained more secreted factors with tissue-regenerating mechanisms, such as cell proliferation, anti-inflammatory effects, and immunomodulatory effects. DPSC-CM was more effective in suppressing the activated T cells than other groups in the flow cytometric analysis. The stimulated salivary flow rate increased in SS mice with DPSC-CM compared with that in the other groups. In addition, the number of inflammation sites in SMGs of the mice administered with DPSC-CM was lower than that in the other groups. The expression levels of interleukin (Il)-10 and transforming growth factor-β1 were upregulated in the DPSC-CM group, whereas those of Il-4 and Il-17a were downregulated. The DPSC-CM-administered group presented with a significantly increased percentage of regulatory T (Treg) cells and a significantly decreased percentage of type 17 Th (Th17) cells compared with the other groups. Conclusions These results indicated that DPSC-CM ameliorated SS by promoting Treg cell differentiation and inhibiting Th17 cell differentiation in the mouse spleen.

scholarly journals Biological and microbiological interactions of Ti-35Nb-7Zr alloy and its basic elements on bone marrow stromal cells: good prospects for bone tissue engineering

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
Daphne de Camargo Reis Mello ◽  
Lais Morandini Rodrigues ◽  
Fabia Zampieri D’Antola Mello ◽  
Thais Fernanda Gonçalves ◽  
Bento Ferreira ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Differentiation ◽  
Stromal Cells ◽  
Biofilm Formation ◽  
Bone Marrow Stromal Cells ◽  
Pure Titanium ◽  
Marrow Stromal Cells ◽  
The Other ◽  
Cell Based Therapy

Abstract Background An effective biomaterial for bone replacement should have properties to avoid bacterial contamination and promote bone formation while inducing rapid cell differentiation simultaneously. Bone marrow stem cells are currently being investigated because of their known potential for differentiation in osteoblast lineage. This makes these cells a good option for stem cell-based therapy. We have aimed to analyze, in vitro, the potential of pure titanium (Ti), Ti-35Nb-7Zr alloy (A), niobium (Nb), and zirconia (Zr) to avoid the microorganisms S. aureus (S.a) and P. aeruginosa (P.a). Furthermore, our objective was to evaluate if the basic elements of Ti-35Nb-7Zr alloy have any influence on bone marrow stromal cells, the source of stem cells, and observe if these metals have properties to induce cell differentiation into osteoblasts. Methods Bone marrow stromal cells (BMSC) were obtained from mice femurs and cultured in osteogenic media without dexamethasone as an external source of cell differentiation. The samples were divided into Ti-35Nb-7Zr alloy (A), pure titanium (Ti), Nb (niobium), and Zr (zirconia) and were characterized by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). After predetermined periods, cell interaction, cytotoxicity, proliferation, and cell differentiation tests were performed. For monotypic biofilm formation, standardized suspensions (106 cells/ml) with the microorganisms S. aureus (S.a) and P. aeruginosa (P.a) were cultured for 24 h on the samples and submitted to an MTT test. Results All samples presented cell proliferation, growth, and spreading. All groups presented cell viability above 70%, but the alloy (A) showed better results, with statistical differences from Nb and Zr samples. Zr expressed higher ALP activity and was statistically different from the other groups (p < 0.05). In contrast, no statistical difference was observed between the samples as regards mineralization nodules. Lower biofilm formation of S.a and P.a. was observed on the Nb samples, with statistical differences from the other samples. Conclusion Our results suggest that the basic elements present in the alloy have osteoinductive characteristics, and Zr has a good influence on bone marrow stromal cell differentiation. We also believe that Nb has the best potential for reducing the formation of microbial biofilms.

In vitro Three Dimensional Scaffold-free Construct of Human Adipose-derived Stem Cells in Coculture with Endothelial Cells and Fibroblasts

2017 ◽  
Vol 68 (6) ◽  
pp. 1341-1344
Author(s):  
Grigore Berea ◽  
Gheorghe Gh. Balan ◽  
Vasile Sandru ◽  
Paul Dan Sirbu
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Endothelial Cells ◽  
Single Cell ◽  
Cell Line ◽  
Bone Repair ◽  
Umbilical Vein ◽  
Human Fibroblasts ◽  
Three Dimensional ◽  
Adipose Derived Stem Cells

Complex interactions between stem cells, vascular cells and fibroblasts represent the substrate of building microenvironment-embedded 3D structures that can be grafted or added to bone substitute scaffolds in tissue engineering or clinical bone repair. Human Adipose-derived Stem Cells (hASCs), human umbilical vein endothelial cells (HUVECs) and normal dermal human fibroblasts (NDHF) can be mixed together in three dimensional scaffold free constructs and their behaviour will emphasize their potential use as seeding points in bone tissue engineering. Various combinations of the aforementioned cell lines were compared to single cell line culture in terms of size, viability and cell proliferation. At 5 weeks, viability dropped for single cell line spheroids while addition of NDHF to hASC maintained the viability at the same level at 5 weeks Fibroblasts addition to the 3D construct of stem cells and endothelial cells improves viability and reduces proliferation as a marker of cell differentiation toward osteogenic line.

Flow Cytometric Analysis of Brain Tumor Stem Cells

2018 ◽  
pp. 69-77
Author(s):  
Minomi K. Subapanditha ◽  
Ashley A. Adile ◽  
Chitra Venugopal ◽  
Sheila K. Singh
Keyword(s):  
Stem Cells ◽  
Brain Tumor ◽  
Flow Cytometric Analysis ◽  
Tumor Stem Cells ◽  
Flow Cytometric ◽  
Brain Tumor Stem Cells

scholarly journals Modulated expression of notch1 during thymocyte development

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 970-976 ◽  
Author(s):  
RP Hasserjian ◽  
JC Aster ◽  
F Davi ◽  
DS Weinberg ◽  
J Sklar
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Flow Cytometric Analysis ◽  
T Cell Differentiation ◽  
Thymocyte Development ◽  
Cd8 Cells ◽  
Flow Cytometric ◽  
Human T Cell ◽  
Weak Staining ◽  
Notch Gene

Abstract The Notch gene family encodes transmembrane proteins that have been implicated in control of diverse cellular differentiation events in the fly, frog, and mouse. Mammalian Notch1 is expressed at high levels in thymus and is mutated in a subset of human T-cell acute lymphoblastic neoplasms, suggesting a role in T-cell differentiation. To investigate the patterns of expression of NOTCH1 protein in thymocytes of the developing and mature thymus, antibodies raised against NOTCH1 were used to perform immunohistochemical and flow cytometric analyses. Strong staining for NOTCH1 within the fetal murine thymus was observed as early as 13.5 days postcoitum. By 17.5 days postcoitum, preferential staining of superficial cortical thymocytes was observed, with weak staining of developing medulla. Flow cytometric analysis and immunohistochemical staining of flow-sorted cells confirmed that the highest levels of NOTCH1 expression in adult murine thymus were present in immature cortical thymocytes (CD24high, CD4-CD8-). In contrast, NOTCH1 expression was low or absent in more mature cortical thymocytes (CD24low, CD4+CD8+), whereas intermediate levels of expression were observed in CD4+CD8- and CD4-CD8+ cells. These data indicate a dynamic pattern of NOTCH1 expression during T-cell differentiation and suggest that downregulation of NOTCH1 may be required for maturation of cortical thymocytes.

scholarly journals Improving the glial differentiation of human Schwann-like adipose-derived stem cells with graphene oxide substrates

2018 ◽  
Vol 8 (3) ◽  
pp. 20180002 ◽  
Author(s):  
Andrea Francesco Verre ◽  
Alessandro Faroni ◽  
Maria Iliut ◽  
Claudio Silva ◽  
Cristopher Muryn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Graphene Oxide ◽  
Peripheral Nerve ◽  
Peripheral Nerve Injury ◽  
Peripheral Nerve Regeneration ◽  
The Other ◽  
Adipose Derived Stem Cells ◽  
Glial Differentiation ◽  
Oxide Substrates

There is urgent need to improve the clinical outcome of peripheral nerve injury. Many efforts are directed towards the fabrication of bioengineered conduits, which could deliver stem cells to the site of injury to promote and guide peripheral nerve regeneration. The aim of this study is to assess whether graphene and related nanomaterials can be useful in the fabrication of such conduits. A comparison is made between graphene oxide (GO) and reduced GO substrates. Our results show that the graphene substrates are highly biocompatible, and the reduced GO substrates are more effective in increasing the gene expression of the biomolecules involved in the regeneration process compared to the other substrates studied.

High-resolution tracking of cell division suggests similar cell cycle kinetics of hematopoietic stem cells stimulated in vitro and in vivo

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 855-862 ◽  
Author(s):  
Robert A. J. Oostendorp ◽  
Julie Audet ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Fetal Liver ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Flow Cytometric ◽  
Differentiation Status ◽  
Kinetics Of

The kinetics of proliferation of primitive murine bone marrow (BM) cells stimulated either in vitro with growth factors (fetal liver tyrosine kinase ligand 3 [FL], Steel factor [SF], and interleukin-11 [IL-11], or hyper–IL-6) or in vivo by factors active in myeloablated recipients were examined. Cells were first labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and then incubated overnight prior to isolating CFSE+ cells. After 2 more days in culture, more than 90% of the in vivo lymphomyeloid repopulating activity was associated with the most fluorescent CFSE+ cells (ie, cells that had not yet divided), although this accounted for only 25% of the repopulating stem cells measured in the CFSE+ “start” population. After a total of 4 days in culture (1 day later), 15-fold more stem cells were detected (ie, 4-fold more than the day 1 input number), and these had become (and thereafter remained) exclusively associated with cells that had divided at least once in vitro. Flow cytometric analysis of CFSE+ cells recovered from the BM of transplanted mice indicated that these cells proliferated slightly faster (up to 5 divisions completed within 2 days and up to 8 divisions completed within 3 days in vivo versus 5 and 7 divisions, respectively, in vitro). FL, SF, and ligands which activate gp130 are thus efficient stimulators of transplantable stem cell self-renewal divisions in vitro. The accompanying failure of these cells to accumulate rapidly indicates important changes in their engraftment potential independent of accompanying changes in their differentiation status.

scholarly journals A Review of Cell-Based Computational Modeling in Cancer Biology

2019 ◽  
pp. 1-13 ◽  
Author(s):  
John Metzcar ◽  
Yafei Wang ◽  
Randy Heiland ◽  
Paul Macklin
Keyword(s):  
Stem Cells ◽  
Computational Modeling ◽  
Single Cell ◽  
Stromal Cells ◽  
Cancer Biology ◽  
Large Scale ◽  
Computational Models ◽  
Three Dimensional ◽  
Discrete Models ◽  
Simulation Studies

Cancer biology involves complex, dynamic interactions between cancer cells and their tissue microenvironments. Single-cell effects are critical drivers of clinical progression. Chemical and mechanical communication between tumor and stromal cells can co-opt normal physiologic processes to promote growth and invasion. Cancer cell heterogeneity increases cancer’s ability to test strategies to adapt to microenvironmental stresses. Hypoxia and treatment can select for cancer stem cells and drive invasion and resistance. Cell-based computational models (also known as discrete models, agent-based models, or individual-based models) simulate individual cells as they interact in virtual tissues, which allows us to explore how single-cell behaviors lead to the dynamics we observe and work to control in cancer systems. In this review, we introduce the broad range of techniques available for cell-based computational modeling. The approaches can range from highly detailed models of just a few cells and their morphologies to millions of simpler cells in three-dimensional tissues. Modeling individual cells allows us to directly translate biologic observations into simulation rules. In many cases, individual cell agents include molecular-scale models. Most models also simulate the transport of oxygen, drugs, and growth factors, which allow us to link cancer development to microenvironmental conditions. We illustrate these methods with examples drawn from cancer hypoxia, angiogenesis, invasion, stem cells, and immunosurveillance. An ecosystem of interoperable cell-based simulation tools is emerging at a time when cloud computing resources make software easier to access and supercomputing resources make large-scale simulation studies possible. As the field develops, we anticipate that high-throughput simulation studies will allow us to rapidly explore the space of biologic possibilities, prescreen new therapeutic strategies, and even re-engineer tumor and stromal cells to bring cancer systems under control.

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