A protocol for generating high numbers of mature and functional human mast cells from peripheral blood

Abstract

This study describes an efficient protocol for generating mature MCs from human peripheral blood with a functional phenotype of connective tissue-type MCs. Use of these cultured human MCs will increase our knowledge and understanding about human MC development and biology in human disease.

Lipid body formation during maturation of human mast cells

“…Fortunately, several protocols for the generation of human MCs have been established ( 23 ), rendering it possible to perform detailed studies on formation, structure, and function of LBs in these cells. We have recently defi ned culture conditions to generate mature and functional human MCs of the connective tissue type from peripheral blood-derived CD34 + progenitors in the presence of SCF ( 24 ). In the present work, we found that differentiation of such CD34 + progenitor cells into mature MCs invariably associates with a steady increase in the number and size of LBs.…”

“…To gain information on the fate of LBs upon MC activation with ensuing exocytosis of the cytoplasmic secretory granules, we compared resting MCs with those having RESULTS To generate mature and functional mast cells, human peripheral blood-derived CD34 + progenitor cells obtained from 18 different donors were cultured under serum-free conditions for up to 15 weeks ( 24 ). To analyze whether intracellular neutral lipid storage sites are formed during the maturation process, the MCs were stained with Oil Red O ( Fig.…”

“…Mature human MCs were generated exactly as described previously ( 24 ). Briefl y, CD34 + progenitor cells were obtained from fresh buffy coats prepared from peripheral blood of healthy by guest, on May 12, 2018 www.jlr.org Downloaded from samples were mounted in fl uorescence mounting medium (DAKO), and z-stacks were captured with a 63× Plan Apo NA 1.40 lens of a LSM 5 DUO confocal laser scanning microscope (Zeiss).…”

Mast cells promote atherosclerosis by inducing both an atherogenic lipid profile and vascular inflammation

“…The mast cell-committed progenitors leave the bone marrow, circulate in the blood as hematopoietic precursor cells [Kitamura and Ito, 2005], and, in the presence of specific chemotactic signals, notably stem cell factor (SCF) and eotaxin [Haley et al, 2000], they adhere to activated endothelial cells via a 4 b 1 integrins, vascular cell adhesion molecule (VCAM-1), and E-selectin [Boyce et al, 2002]. Upon activation of chemokine receptors, notably CXCR2, CCR3, CXCR4, and CCR5 [Ochi et al, 1999], the precursor cells migrate into tissues and differentiate into mature mast cells in the presence of appropriate growth factors, including SCF, interleukin-3 (IL-3), IL-4, IL-6, IL-9, and nerve growth factor (NGF) [Saito et al, 1996;Kinoshita et al, 1999;Kanbe et al, 2000;Matsuzawa et al, 2003;Lappalainen et al, 2007]. The crucial requirement of a functional SCF and c-kit signaling system for mast cell growth and development is emphasized by the fact that lack of c-kit signaling in mice results in mast cell deficiency [Kitamura et al, 1978;Chabot et al, 1988;Copeland et al, 1990], whereas an elevated expression of c-kit in humans induces mastocytosis [Castells et al, 1996].…”

CCR1-Mediated STAT3 Tyrosine Phosphorylation and CXCL8 Expression in THP-1 Macrophage-like Cells Involve Pertussis Toxin-Insensitive Gα14/16 Signaling and IL-6 Release

“…THP-1 and U-937 cells were differentiated into macrophage-like cells by treatment with 20 nM PMA for 48 h. HEK293 cells were cultured in MEM supplemented with 10% FBS, 50 U/ml penicillin, and 50 mg/ml streptomycin. Primary human mast cell cultures were prepared from peripheral blood CD34 + progenitor using a modified protocol of Lappalainen et al (38). Briefly, fresh peripheral blood buffy coat of healthy adult donors was provided by the Hong Kong Red Cross.…”