Differentiation of mast cell subpopulations from mouse embryonic stem cells

Abstract

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 activation-induced survival.

Introduction

Mast cells act as sentinels during both innate and adaptive immune responses and rapidly respond to exogenous stimuli (Abraham and St John, 2010, Dawicki and Marshall, 2007, Mekori and Metcalfe, 2000, Sayed et al., 2008). A new picture of mast cell function has emerged where these cells have the capacity to both enhance and later help to limit certain innate and adaptive immune responses. Understanding how these positive and negative functions can be induced and suppressed in various mast cell populations is of high interest.

Mast cells are not a homogenous population of cells. In rodents, mast cells are divided into two main subtypes: connective tissue mast cells (CTMC) and mucosal mast cells (MMC) which differ in size, histamine content and neutral protease and proteoglycan composition (Enerback, 1966, Metcalfe et al., 1997). Depending on the influence of the surrounding tissue microenvironment, mast cells can have different phenotypes and functions, even in the same tissue (Williams et al., 2000). The number of mast cells in connective tissue is normally relatively constant, whereas mast cell numbers in the gut, respiratory and gastrointestinal tract can differ considerably. During acute or chronic inflammation mast cell numbers will increase in the affected tissue (Nilsson et al., 1999).

The two mast cell subtypes, CTMC and MMC, are known to respond differently to various well known stimuli. Their reactivity to the mast cell secretagogue compound 48/80 diverges with degranulation occurring in CTMC but not MMC (Enerback and Lowhagen, 1979, Gomez et al., 1987, Stanovnik et al., 1988). In addition, CTMC are sensitive to stimulation by the neuropeptide substance P while MMC are not (Amano et al., 1998, Ogasawara et al., 1997). Upon IgE receptor cross-linking mast cells of CTMC like type but not MMC like type induce a survival program, upregulating the pro-survival Bcl-2 family member A1 that enhances their survival after growth factor deprivation (Ekoff et al., 2007). Beyond distinct activation patterns, the two mast cell subtypes also vary in proliferation rate (Ekoff et al., 2007). Due to their differences, generalization of findings from single mast cell populations could be problematic.

As the spectrum of diseases that involves mast cells increases, so do the questions concerning what diverse triggers that could affect mast cell reactivity. Apart from the classical IgE-dependent inflammatory processes, mast cells can be activated by endogenous stimuli like complement factors, neuropeptides and hormones and exogenous factors like pathogens, drugs and toxins (Dawicki et al., 2007). Dependent on the population of mast cells examined, however, their sensitivity to activation by various stimuli differs. The susceptibility of mast cells to activation by numerous compounds and their capacity to react uniquely to a range of stimuli, with release of potent factors, with or without degranulation, make them powerful cells both in health and disease.

Previous studies of mast cell development into specific phenotypes have mainly been performed on bone marrow derived mast cells. To reduce the use and handling of experimental animals, however, differentiation of mast cell from ES cells is an alternative method. In addition, derivation of mast cells from ES cells enables the generation of mast cells from mice that carry genetic modifications that are embryonic lethal e.g., bcl-x knock out mice (Moller et al., 2007). The ES derived cell system could further be used to screen for the effect, toxicity and specificity of different drugs and compounds for one or both mast cell subtypes.

In this study we describe in vitro development of specific subtypes, MLMC and CTLMC, from ES cells. The mast cell populations were characterized by staining patterns, protease content, cell surface receptor expression and activation-induced survival.