Issan Yee San Tam1, Chun Wai Ng1, See-Ying Tam2, Hang Yung Alaster Lau3. 1. School of Biomedical Sciences, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. 2. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. stam@stanford.edu. 3. School of Biomedical Sciences, Faculty of Medicine, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong. hyalau@cuhk.edu.hk.
Abstract
OBJECTIVE: The aim of this study was to develop a novel protocol for generating large populations of fully mature and functional human mast cells (HMC) from CD34+ hematopoietic stem cells which require less culturing time than previously reported methods. METHODS: CD34+ cells isolated from fresh human buffy coats were sequentially cultured with different combinations of SCF, IL-6, IL-3, IL-9 and IL-4 under selected culturing conditions and time periods. Cells were then harvested for immunohistochemical characterization of morphological phenotypes and were functionally characterized by assessing their responses to IgE-dependent and -independent stimuli by measuring the release of inflammatory mediators and cytokines. Moreover, the pharmacological profiles of several classes of anti-inflammatory drugs in inhibiting the activation of these HMC were also characterized. RESULTS: We have developed a novel protocol that can generate large homogenous populations of mature and functional HMC in 6 weeks. These cells expressed both tryptase and chymase and were activated by anti-IgE, cationic peptides and calcium ionophores. Moreover, IgE-dependent activation of these cells was significantly inhibited by anti-inflammatory drugs. The morphological and functional characteristics of these mast cells resembled those of MCTC type or connective tissue-type HMC. DISCUSSION: Our protocol represents a novel time-saving and economical approach for generating large numbers of primary HMC for functional studies of mast cell biology and for profiling novel anti-inflammatory therapeutic agents with mast cell-inhibitory properties in humans.
OBJECTIVE: The aim of this study was to develop a novel protocol for generating large populations of fully mature and functional human mast cells (HMC) from CD34+ hematopoietic stem cells which require less culturing time than previously reported methods. METHODS:CD34+ cells isolated from fresh human buffy coats were sequentially cultured with different combinations of SCF, IL-6, IL-3, IL-9 and IL-4 under selected culturing conditions and time periods. Cells were then harvested for immunohistochemical characterization of morphological phenotypes and were functionally characterized by assessing their responses to IgE-dependent and -independent stimuli by measuring the release of inflammatory mediators and cytokines. Moreover, the pharmacological profiles of several classes of anti-inflammatory drugs in inhibiting the activation of these HMC were also characterized. RESULTS: We have developed a novel protocol that can generate large homogenous populations of mature and functional HMC in 6 weeks. These cells expressed both tryptase and chymase and were activated by anti-IgE, cationic peptides and calcium ionophores. Moreover, IgE-dependent activation of these cells was significantly inhibited by anti-inflammatory drugs. The morphological and functional characteristics of these mast cells resembled those of MCTC type or connective tissue-type HMC. DISCUSSION: Our protocol represents a novel time-saving and economical approach for generating large numbers of primary HMC for functional studies of mast cell biology and for profiling novel anti-inflammatory therapeutic agents with mast cell-inhibitory properties in humans.
Entities:
Keywords:
Allergy; Buffy coat; Culture protocol; Human mast cell; Inflammation
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