Generation and utilization of a HEK-293T murine GM-CSF expressing cell line.

Elektra Kantzari Robinson,Susan Carpenter,Simon Zhou,Sergio Covarrubias,Donna A Macduff

doi:10.1371/journal.pone.0249117

Elektra Kantzari Robinson, Susan Carpenter + Show 3 more

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https://doi.org/10.1371/journal.pone.0249117

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Journal: PloS one	Publication Date: Apr 9, 2021
Citations: 3	License type: CC BY 4.0

Affiliation: University of California, Santa Cruz

Abstract

Macrophages and dendritic cells (DCs) are innate immune cells that play a key role in defense against pathogens. In vitro cultures of bone marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) are well-established and valuable methods for immunological studies. Typically, commercially available recombinant GM-CSF is utilized to generate BMDCs and is also used to culture alveolar macrophages. We have generated a new HEK-293T cell line expressing murine GM-CSF that secretes high levels of GM-CSF (~180 ng/ml) into complete media as an alternative to commercial GM-CSF. Differentiation of dendritic cells and expression of various markers were kinetically assessed using the GM-CSF HEK293T cell line, termed supGM-CSF and compared directly to purified commercial GMCSF. After 7-9 days of cell culture the supGM-CSF yielded twice as many viable cells compared to the commercial purified GM-CSF. In addition to differentiating BMDCs, the supGM-CSF can be utilized to culture functionally active alveolar macrophages. Collectively, our results show that supernatant from our GM-CSF HEK293T cell line supports the differentiation of mouse BMDCs or alveolar macrophage culturing, providing an economical alternative to purified GM-CSF.

Highlights

Colony-stimulating factors (CSF) including macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF known as colony stimulating factor 2, CSF2) are crucial for survival, proliferation, differentiation and functional activation of hematopoietic cells, including macrophages and dendritic cells (DCs) [1]
HEK-293T murine Granulocyte-macrophage colony-stimulating factor (GM-CSF) cell line differentiation, it is appreciated that Flt3L DCs are representative of steady-state resident DCs, while GM-CSF BMDCs mirror the transcriptional programing of pro-inflammatory recruited cells [22, 23]
Granulocyte-macrophage colony-stimulating factor (GM-CSF), differentiated bone marrow cells are widely used as a model system for conventional DC development [24, 25], as well as sustaining primary alveolar macrophages in culture [26]

Summary

Introduction

Colony-stimulating factors (CSF) including macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony stimulating factor (GM-CSF known as colony stimulating factor 2, CSF2) are crucial for survival, proliferation, differentiation and functional activation of hematopoietic cells, including macrophages and dendritic cells (DCs) [1]. Macrophages and DCs are innate immune cells found in tissues and lymphoid organs that play a key role in defense against pathogens [2]. While there are a multitude of macrophage and dendritic cell subsets, GM-CSF is critical for the development of conventional dendritic cells (cDCs) and alveolar macrophages (AMs) [2]. Due to cell number limitations from harvesting cDCs and AMs directly from mice, well-established in vitro culturing of bone marrow and bronchoalveolar lavage fluid for dendritic cells and alveolar macrophages, respectively, using GM-CSF have become invaluable for immunological.

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