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Jeddi, Marjan (2020) Identification of a novel method for differentiating human monocytic cell line into macrophages. Masters thesis, London Metropolitan University.
Introduction:
Monocytes and macrophages are essential for optimum function of the natural immune response from recognising and killing infection, to body repair and signal transfer between cells. Macrophages within tissues are specialised macrophages and adapt their structure and function to optimise fighting foreign material and infections. Part of the immune response involves release of microvesicles (MVs) formed by the outward budding of the plasma membrane supporting intercellular communication. This research developed a method for producing macrophage-like cells under conditions that may stimulate in vivo using a monocytic cell (THP-1) and a human liver cell (HUH7). The basis for choosing these two cells stemmed from research by Matak et al confirming their cellular communication.
Aim:
The aim of the research was to compare two different methods of THP-1 monocyte differentiation into M0 macrophages. An In vitro protocol was established using phorbol 12-myristate-13-acetate (PMA) and was compared to a conditioned medium model using THP-1 and HUH7 hepatoma cells.
Method:
THP-1 cells were incubated with 100nM of PMA for 72 hours to differentiate into M0 macrophage-like cells. The novel method used THP-1 cells incubated with HUH7 conditioned medium and monitored over 120 hours to allow differentiation and confluent cell growth.
Results:
THP-1 cells differentiated into M0 macrophage-like cells changing morphology from rounded to elongated cells with the characteristic macrophage phenotype. THP-1 cells with a conditioned medium produced a permanent macrophage like cell line named THP-H.
Conclusion:
THP-1 cells differentiated into macrophages using two different methods; an established method of 100nM of PMA over 72 hours and with a novel method of HUH7- conditioned cell medium. However, PMA is a toxic artificial reagent used for differentiating THP-1 cells thus, produced short-lived macrophages. The novel method produced a permanent macrophage cell line, THP-H, with similar physiological conditions for replicating the study in vivo. Cell communication, possibly assisted by MVs, occurs between THP-1 and HUH7 cells to produce a novel permanently differentiated macrophage like cell. This novel research has therefore identified a cross-talk between THP-1 and HUH7 cells. Furthermore, these findings can be extended to research the known effects of activated macrophages in inducing hepcidin expression in HUH7 cells, by investigating the signals transferred by EVs between the two cells. Moreover, this research can provide the basis for uncovering mechanisms of communication between the THP-1 and HUH7 cells for cancer diagnosis, cell markers for cell differentiation and a new cell line named THP-H can be fully characterised.
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