Abstract
SummaryHuman induced pluripotent stem cells (iPSCs) and macrophages derived from them are increasingly popular tools for research into both infectious and degenerative diseases. However, as the field strives for greater modeling accuracy, it is becoming ever more challenging to justify the use of undefined and proprietary media for the culture of these cells. Here, we describe a defined, serum-free, open-source medium for the differentiation of iPSC-derived macrophages. This medium is equally capable of maintaining these cells compared with commercial alternatives. The macrophages differentiated in this medium display improved terminally differentiated cell characteristics, reduced basal expression of induced antiviral response genes, and improved polarization capacity. We conclude that cells cultured in this medium are an appropriate and malleable model for tissue-resident macrophages, on which future differentiation techniques can be built.
Highlights
Resident phagocytes are an evolutionarily conserved cell type in metazoans
To investigate the molecular pathways involved in the pathogenesis of both infectious diseases, such as those caused by human immunodeficiency virus-1 (HIV-1) and Mycobacterium tuberculosis (Hartlova et al, 2018), and degenerative diseases, especially Parkinson disease (Haenseler et al, 2017a; Lee et al, 2020) and Alzheimer disease (Brownjohn et al, 2018; Garcia-Reitboeck et al, 2018), we developed a pathophysiologically authentic, yet genetically tractable, model of human tissue macrophages derived from pluripotent stem cells (Karlsson et al, 2008; van Wilgenburg et al, 2013)
Our first method involved spontaneous differentiation of mesoderm from embryonic stem cells via embryoid body (EB), followed by myeloid differentiation using macrophage colony stimulating factor (M-CSF) (CSF-1) and interleukin-3 (IL-3) in a serum-supplemented medium (Karlsson et al, 2008). Since this was not effective for all pluripotent stem cell lines, and to remove the undefined serum component, we subsequently developed a serumfree method, using BMP4, VEGF, and SCF to promote mesodermal lineage and prime hemogenic endothelium differentiation during EB formation, and the serum-free X-VIVO 15 (XVIVO) medium (Lonza) during the M-CSF/ IL-3-directed myeloid differentiation stage
Summary
Resident phagocytes are an evolutionarily conserved cell type in metazoans. In mammals, resident macrophages support tissue homeostasis through a wide range of specialized trophic, remodeling, and defense functions, whose importance is illustrated by their failure in malignant, degenerative, and infectious diseases (Gordon et al, 2014; Steinman and Moberg, 1994). Numerous methods for the differentiation of macrophages from stem cells have been developed over the years, building upon advances in our understanding of the requirements for hematopoiesis (Rajab et al, 2018; Rowe et al, 2016) These can be broadly categorized into three types: monolayer cultures, co-cultures with xeno cells, and via embryoid body (EB) intermediates. Our first method involved spontaneous differentiation of mesoderm from embryonic stem cells via EBs, followed by myeloid differentiation using macrophage colony stimulating factor (M-CSF) (CSF-1) and interleukin-3 (IL-3) in a serum-supplemented medium (Karlsson et al, 2008) Since this was not effective for all pluripotent stem cell lines, and to remove the undefined serum component, we subsequently developed a serumfree method, using BMP4, VEGF, and SCF to promote mesodermal lineage and prime hemogenic endothelium differentiation during EB formation, and the serum-free X-VIVO 15 (XVIVO) medium (Lonza) during the M-CSF/ IL-3-directed myeloid differentiation stage (van Wilgenburg et al, 2013)
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