Generation Of Human Macrophages Research Articles

Alveolar macrophages in tissue homeostasis, inflammation, and infection: evolving concepts of therapeutic targeting.

Alveolar macrophages (AMs) are the sentinel cells of the alveolar space, maintaining homeostasis, fending off pathogens, and controlling lung inflammation. During acute lung injury, AMs orchestrate the initiation and resolution of inflammation in order to ultimately restore homeostasis. This central role in acute lung inflammation makes AMs attractive targets for therapeutic interventions. Single-cell RNA-Seq and spatial omics approaches, together with methodological advances such as the generation of human macrophages from pluripotent stem cells, have increased understanding of the ontogeny, function, and plasticity of AMs during infectious and sterile lung inflammation, which could move the field closer to clinical application. However, proresolution phenotypes might conflict with proinflammatory and antibacterial responses. Therefore, therapeutic targeting of AMs at vulnerable time points over the course of infectious lung injury might harbor the risk of serious side effects, such as loss of antibacterial host defense capacity. Thus, the identification of key signaling hubs that determine functional fate decisions in AMs is of the utmost importance to harness their therapeutic potential.

IPSC-Derived Macrophages: The Differentiation Protocol Affects Cell Immune Characteristics and Differentiation Trajectories.

The generation of human macrophages from induced pluripotent stem cells (iMacs) is a rapidly developing approach used to create disease models, screen drugs, study macrophage-pathogen interactions and develop macrophage-based cell therapy. To generate iMacs, different types of protocols have been suggested, all thought to result in the generation of similar iMac populations. However, direct comparison of iMacs generated using different protocols has not been performed. We have compared the productivity, the differentiation trajectories and the characteristics of iMacs generated using two widely used protocols: one based on the formation of embryoid bodies and the induction of myeloid differentiation by only two cytokines, interleukin-3 and macrophage colony-stimulating factor, and the other utilizing multiple exogenous factors for iMac generation. We report inter-protocol differences in the following: (i) protocol productivity; (ii) dynamic changes in the expression of genes related to inflammation and lipid homeostasis following iMac differentiation and (iii) the transcriptomic profiles of terminally differentiated iMacs, including the expression of genes involved in inflammatory response, antigen presentation and lipid homeostasis. The results document the dependence of fine iMac characteristics on the type of differentiation protocol, which is important for further development of the field, including the development of iMac-based cell therapy.

Increasing the yield of human mononuclear cells and low serum conditions for in vitro generation of macrophages with M-CSF

The yield of mononuclear cells extracted from peripheral blood using standard protocols is frequently inadequate when working with material of limited availability. In addition, the regular usage of autologous and fetal calf serum (FCS) to generate human macrophages in vitro may complicate antigen uptake, processing and presentation on HLA molecules. We optimized the yield of mononuclear cells from 34±3% to 65±5% by collecting both the interface and more than half of the overlayering supernatant, followed by three washes at 4 °C. Monocytes from 12 individuals were cultured 1–4 days with 0–100 ng/ml macrophage colony-stimulating factor (M-CSF) at either 1% (low) or 5% (v/v) FCS. Regardless of number of days in culture, maximal (50–100 ng/ml) M-CSF stimulation and low FCS induced 65±5% esterase-positive cells in all individuals compared to 52±7% without M-CSF ( p<0.001). M-CSF increased the mean proportion of esterase-positive cells in both 1% and 5% FCS with a negative interaction found between 5% FCS and M-CSF ( p<0.05). All cells were positive for CD14 and HLA class II but cell number did not increase. The generation of human macrophages by M-CSF at low FCS should prove useful in studies where higher FCS concentrations may interfere with the assay.