Efficient, Long Term Production of Monocyte-Derived Macrophages from Human Pluripotent Stem Cells under Partly-Defined and Fully-Defined Conditions

Abstract

Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively, up to ∼3×107 monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14+, CD16low, CD163+). Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.

Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis

“…We here applied a modified version of a protocol established by van Wilgenburg and coworkers (26), which allows highly reproducible production of large numbers of monocyte-and macrophage-like cells from individual EBs over 60 days and more. This is in contrast to the situation for reconstituting HSCs.…”

“…iPSC lines were subjected to a modified version of the embryoid body (EB)-based hematopoietic differentiation protocol described by van Wilgenberg and coworkers (26). In brief, pluripotent stem cell lines were diluted to single cells and cultured for 5 days in ESC-medium in six-well suspension plates on an orbital shaker (100 rpm) to form embryoid bodies.…”

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

“…The hPSC H1 cell line was used to generate the PB-CRISPR/Cas hPSCs by piggyBac transposon-mediated gene engineering and subsequently differentiated into monocyte/macrophages through spin-EB generation using a feeder-free protocol, which was modified from previous publications 27,28 . Cells form spin EBs on 96-well low attachment plates supplemented with cytokines (SCF, VEGF and BMP4) and were grown for 4 days.…”

“…4d). These CRISPR/Cas9-hPSCs can be differentiated into monocytes/macrophages using a feeder-free protocol 27,28 ( Fig. 4e).…”

FRMD8 promotes inflammatory and growth factor signalling by stabilising the iRhom/ADAM17 sheddase complex

“…For this study 563 iPSCs were differentiated into embryoid bodies (EBs) by mechanical lifting of iPSC colonies 564 and differentiated into macrophages as described in (van Wilgenburg et al, 2013). Briefly, 565 iPSCs were grown on a feeder layer of MEFs in hES medium.…”