THE IN VITRO DIFFERENTIATION OF MAST CELLS: Cultures of Cells from Immunized Mouse Lymph Nodes and Thoracic Duct Lymph on Fibroblast Monolayers (original) (raw)
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The in vitro differentiation of mast cells
The Journal of Cell Biology
When cells from lymph nodes or thoracic duct of mice hyperimmunized with protein antigens are cultivated on embryo monolayers in the presence of the antigen, numerous clones of mast cells appear. The histochemical and ultrastructural characteristics of the cells permit their identification as mast cells and distinguish them from the phagocytic histiocytes that usually arise in abundance in similar cultures from unimmunized mouse cells or from immunized mouse cells cultured in the absence of the antigen. Only a few colonies of mast cells appeared in the latter cultures. The basis for the induction of mast cell differentiation is not known.
Differentiation and proliferation of embryonic mast cells of the rat
The Journal of Cell Biology
Histochemical reactions and radioautography were used to investigate the sequence of mast cell development in rat embryos. Mast cells arise ubiquitously in and are confined to the loose connective tissue in the embryo. The alcian blue--safranin reaction distinguishes between weakly sulfated and strongly sulfated mucopolysaccharides by a shift from alcian blue to safranin staining. Based on this reaction and morphologic characteristics, four stages were identified. Stage I mast cells are lymphocyte-like cells with cytoplasmic granules which invariably stain blue with the alcian blue-safranin reaction. In Stage II cells the majority of granules are alcian blue-positive, but some safranin-positive granules have appeared. Stage III mast cells are distinguished by a majority of safranin-positive cytoplasmic granules; some alcian blue-posltive granules still remain. Stage IV cells contain only safranin-positive granules. Thymidine-H 3 uptake and identification of mitotic figures indicates that mast cells in Stages I and II comprise a mitotic pool while those in Stages III and IV are mitotically inactive. The pattern of $3~)4 incorporation and the sequence of appearance of histochemically identifiable mast cell constituents corroborates division of the proliferation and differentiation of embryonic mast cells into the four stages described above. The process of formation of mast cell granules is interpreted as reflecting the synthesis and accumulation of a heparin precursor in alcian blue positive granules followed by the synthesis and accumulation of highly N-sulfated heparin along with mast cell chymase and finally histamine in safranin-positive granules.
Journal of Histochemistry & Cytochemistry, 2001
Previous studies of mast cell maturation, structure, and function have been hampered by the lack of mast cell-specific markers. In this study, using a well-characterized mast cell-specific monoclonal antibody, MAb AA4, mast cells from rat bone marrow in various stages of maturation were isolated and characterized. The very immature mast cells, which have not been previously described, contained few granules and would not be recognized as mast cells by standard cytological methods. Pure populations of mast cells were isolated from the bone marrow using MAb AA4-conjugated magnetic beads. The same stages of maturation were observed in the isolated mast cells as were seen in the unfractionated bone marrow. All of these cells were immunopositive for the ␣ -subunit of Fc RI, IgE, and c-kit, confirming their identity as mast cells. By direct counting of immunolabled cells and by flow cytometry, approximately 2.4% of the cells in the bone marrow are mast cells. Staining with toluidine blue and berberine sulfate, as well as RT-PCR of the cells, indicates that these cells are connective tissue-type mast cells. The use of immunological methods for identification of mast cell precursors should facilitate the study of these cells.
Journal of Immunological Methods, 1992
chlhcr It)t)l. itcccplctl 13 December PAt,H) A simple method is described for the preparation of large numbcrs of mast cells from mouse spleen cells in vitro. Mouse spleen cells were cultured with RPMI 1640 medium supplemented with 10% FCS and 2-ME. Half of the total volume of the medium was changed every 4-5 days. Mast cell numbers increased with the culture time and reached a peak between 16 and 2(1 days. Using this method, 2 × I(1" mast cells could be induced from I × 107 nucleated normal spleen cells. T cells and supernatant derived from ConA-stimulated T cells were unnecessary for mast cell induction. Phenotype analysis by FACS showed that Thyl,2, L3T4, Ly-2, Ig, B220, Asialo GM~, and WGA receptors were all negative but functional igE receptors were positive. The granules in the cells could be stained by alcian blue but not by safranin. There was 1.632 + 0.024 ~g stored histamine in 1 × 10" of the cells. Histamine was released from the cells in an antigen-induced and lgE-mcdiated proccss. Compound 48/80 and A23187 induced degranulation of the cells, and the mast cells were able to respond to ConA.
Development of human mast cells in vitro
Proceedings of the National Academy of Sciences, 1989
Nucleated cells of human umbilical cord blood were cocultured with mouse skin-derived 3T3 fibroblasts. After 7-8 weeks in culture, when the number of the other hematopoietic cells declined, metachromatic granule-containing mononuclear cells appeared in the cultures, and the number of the cells increased up to 12 weeks. After 11-14 weeks in culture, the metachromatic mononuclear cells comprised a substantial portion of the cultured cells. These cells contained 1.8-2 micrograms of histamine per 10(6) cells and bore receptors for IgE. All of the cells contained tryptase in their granules. Electron microscopic analysis showed that these cells were mature human mast cells, clearly different from the basophilic granulocytes or eosinophils that arise in a variety of circumstances in cord blood cell cultures. Most of the cultured mast cells expressed some granules with regular crystalline arrays and contained both tryptase and chymase, and thus resembled human skin mast cells.
Characterization of rat tissue cultured mast cells
European Journal of Immunology, 1990
Twelve continuous rat tissue cultured mast cell (MC) lines were established by prolonged culture of rat peritoneal MC in the absence of added factors or feeder layers. Two of these lines, RCMCl and RCMC2, have been briefly described previously, seven others are now also described. Both RCMCl and RCMC2 lack a marker chromosomes present on RBL-CA10.7 cells. All lines were found to express the phenotype of mucosal MC as defined by alcian blue-positive and safranin 0-negative staining, the presence of rat MC protease I1 and a low histamine content. When analyzed for high-(FcsRI) and low-affinity (FcsRL) receptors for IgE, the various lines yielded a variety of receptor patterns. Northern blot analysis of the RNA of RCMCl, RCMC2 and RBL-CA10.7 revealed that all three cell lines contained the same mRNA species for the a , p and y subunits for FcsRI previously found in another rat basophilic leukemia cell line. Quantitation of the relative amounts of a , p and y mRNA did not correlate with the expression of the relative amounts of FceRI(a) in these cells.The relative amounts of mRNA for all these subunits of RCMC2 were equal or higher than those of RCMCl, suggesting that the low expression of FcsRI(a) on the former was a consequence of post-transcriptional events. Analysis of a RCMCl clone over a 6-month period revealed changes in the expression of both FceRI(a) and FCERL. Abbreviations: MC: Mast cell CTMC: Connective tissue MC FcERI: High-affinity receptor for IgE FCERL: Low-affinity receptor for IgE, previously named H or FcERII. The latter designation was discontinued to avoid confusion with FcERII or CD23 found on B cells MMC: Mucosal MC RBL: Rat basophilic leukemia RCMC: Rat tissue cultured MC RMCP: Rat MC protease R(M)pMC: Rat (mouse) peritoneal MC TfR: Transferrin receptor 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
Origin, maturation and recruitment of mast cell precursors
Frontiers in Bioscience, 2011
Introduction 3. Origin of mast cell committed precursors 3.1. Mouse bone marrow derived mast cell committed precursor 3.2. Human bone marrow derived mast cell committed precursor 4. Mast cell maturation 4.1. Maturation of bone marrow derived mast cells 4.2. Maturation of mast cells at peripheral sites 4.3. Major factors affecting mast cell proliferation and maturation 5. Mast cell recruitment 6. Conclusions 7. References
Differentiation of mast cell subpopulations from mouse embryonic stem cells
Journal of Immunological Methods, 2012
Mast cells can generally be divided into two major groups, connective tissue mast cells and mucosal mast cells. We and others have previously shown that these mast cell populations can be developed in vitro from mouse bone marrow stem cells using a combination of specific growth factors and cytokines. Mast cell differentiation from mouse embryonic stem (ES) cells is an important alternative method when developing mast cells from an embryonic lethal genetic deficiency or to reduce the use and handling of experimental animals. In this study, we have used protocols prior known to induce connective tissue like mast cells (CTLMC) (SCF and IL-4) and mucosal like mast cells (MLMC) (SCF, IL-3, IL-9 and TGF-β) from mouse bone marrow progenitor cells and employed these protocols to study if phenotype specific mast cells can be developed from ES cells. We here demonstrate that mast cells of the different phenotypes, CTLMC and MLMC, can be derived from mouse ES cells. The mast cell populations were characterized by chymase expression, receptor expression and their difference in activation pattern and in activationinduced survival.