Abstract
Mast cells (MCs) arise from hematopoietic stem cells (HSCs) that mature within vascularized tissues. Fibroblasts and endothelial cells (ECs) play a role in the maturation of HSCs in the tissues. Due to difficulties in isolating MCs from tissues, large numbers of committed MC precursors can be generated in 2D culture systems with the use of differentiation factors. Since MCs are tissue-resident cells, the development of a 3D tissue-engineered model with ancillary cells that more closely mimics the 3D in vivo microenvironment has greater relevance for MC studies. The goals of this study were to show that MCs can be derived from HSCs within a 3D matrix and to determine a media to support MCs, fibroblasts, and ECs. The results show that HSCs within a collagen matrix cultured in StemSpan media with serum added at the last week yielded a greater number of c-kit+ cells and a greater amount of histamine granules compared to other media tested. Media supplemented with serum were necessary for EC survival, while fibroblasts survived irrespective of serum with higher cell yields in StemSpan. This work demonstrates the development of functional MCs within a 3D collagen matrix using a stem cell media that supports fibroblast and ECs.
Highlights
Release of preformed mediators and expression of diverse molecules have placed mast cells (MCs) among the foremost inducers of allergic responses and regulators of innate and adaptive immunity [1, 2]
M199, our standard media for endothelial cells (ECs) culture that was used for fibroblasts, either with serum added from the beginning or in the last week of culture, did not support MC generation and survival, as verified by microscopy, viability, and flow cytometry analyses
We have established that MCs can be generated from hematopoietic stem cells (HSCs) isolated from peripheral blood within a 3D collagen matrix, based on the morphology of the CD133+-derived cells, the formation of cytoplasmic granules, and the expression of MC phenotypic markers
Summary
Release of preformed mediators and expression of diverse molecules have placed mast cells (MCs) among the foremost inducers of allergic responses and regulators of innate and adaptive immunity [1, 2]. MCs are abundant in tissue near surfaces exposed to the external environment, and their number and distribution change markedly during immune responses [3,4,5]. MCs are not normally present in circulation, they can be obtained from progenitor cells in the presence of T cell-derived cytokines and fibroblast-derived stem cell factor (SCF) [10,11,12]. CD133 and CD34 antigens are markers for primitive progenitor and hematopoietic stem cell (HSC) populations [13, 14]. Previous studies have shown that upon treatment with SCF, interleukin- (IL-) 3, and IL-6, CD133+
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