Abstract
Bone-marrow-derived mast cells are matured from bone marrow cells in medium containing 20% fetal calf serum (FCS), interleukin (IL)-3 and stem-cell factor (SCF) and are used as in vitro models to study mast cells (MC) and their role in health and disease. In vivo, however, BM-derived hematopoietic stem cells account for only a fraction of MC; the majority of MC in vivo are and remain tissue resident. In this study we established a side-by-side culture with BMMC, fetal skin MC (FSMC) or fetal liver MC (FLMC) for comparative studies to identify the best surrogates for mature connective tissue MC (CTMC). All three MC types showed comparable morphology by histology and MC phenotype by flow cytometry. Heterogeneity was detected in the transcriptome with the most differentially expressed genes in FSMC compared to BMMC being Hdc and Tpsb2. Expression of ST2 was highly expressed in BMMC and FSMC and reduced in FLMC, diminishing their secretion of type 2 cytokines. Higher granule content, stronger response to FcεRI activation and significantly higher release of histamine from FSMC compared to FLMC and BMMC indicated differences in MC development in vitro dependent on the tissue of origin. Thus, tissues of origin imprint MC precursor cells to acquire distinct phenotypes and signatures despite identical culture conditions. Fetal-derived MC resemble mature CTMC, with FSMC being the most developed.
Highlights
Mast cells (MC) are tissue-resident immune cells that play a crucial role in different types of immune responses [1]
We describe the generation of MC originating from bone-marrow (BMMC), fetal liver (FLMC), and fetal skin (FSMC) and compare these three MC types regarding phenotypic markers, proliferation, and mediator content as well as response to mitotic activation, IgE-crosslinking of the FcεRI receptor and innate stimulation
Bone marrow cells were prepared from femur and tibia and cultured in RPMI 1640 medium (Biochrom, Berlin, Germany) with 20% fetal calf serum (FCS) (CH 30160.03 Hyclone Perbio), 1% X63Ag8-653mIL-3–conditioned medium corresponding to 10 ng IL-3/mL, 1% CHO–murine stem cell factor conditioned media corresponding to 50 ng mSCF/mL), 50 U/mL penicillin/streptomycin (Biochrom, Berlin, Germany) and μM β-mercaptoethanol (Merck, Darmstadt, Germany), for days at 37 ◦C and 5% CO2
Summary
Mast cells (MC) are tissue-resident immune cells that play a crucial role in different types of immune responses [1]. Contrary to the classic tree-like model describing hematopoiesis, single-cell RNA sequencing studies suggest that hematopoietic stem cells and progenitor cells differentiate along trajectories [4]. MC progenitors (MCP) migrate into target tissues during their maturation, i.e., the intestine, skin, or serosal cavity, by extravasating from the bloodstream under basal conditions dependent on trafficking molecules CXCR2 and ß7 integrin and chemoattractant gradients [6,7]. Their preferential localization in the host–environmental interface allows MC to act as sentinels for tissue damage and pathogen invasion [8]. The process of becoming mature MC following tissue infiltration as progenitor cells is controlled by the microenvironment of the tissue providing stimuli such as IL-3, that lead to maturation of MC [9]
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