Induced Pluripotent Stem Cells Derived Mesenchymal Stem Cells and Macrophages Interplay to Promote Tissue Repair

Zhang, Jiandong (2025) Induced Pluripotent Stem Cells Derived Mesenchymal Stem Cells and Macrophages Interplay to Promote Tissue Repair. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Macrophage polarisation into the pro-inflammatory M1 phenotype and the anti-inflammatory M2 phenotype plays a crucial role in inflammation, resolution, and tissue regeneration. It was well established that lipopolysaccharide (LPS) directed macrophages toward M1 polarisation. It is well established that lipopolysaccharide (LPS) directs macrophages toward M1 polarization. In contrast, interleukin-4 (IL-4) and interleukin-13 (IL-13) promote M2 polarization; however, the underlying mechanisms remain incompletely understood. Mesenchymal stem cells (MSCs) were known to modulate macrophage function and promote an anti-inflammatory phenotype, but how they influenced polarisation at the molecular level had yet to be fully elucidated. This study initially carried out RNA deep sequencing (RNA-Seq) on macrophages and, for the first time, discovered that LPS suppressed the expression of the Mer proto-oncogene tyrosine kinase (MerTK) gene in macrophages. This finding was validated at the transcriptional level using RT-qPCR and at the protein level through western blotting and confocal microscopy. Furthermore, inhibition of NF-κB restored MerTK expression in response to LPS, as confirmed by RT-qPCR. Promoter analysis identified an E-box motif within the MerTK promoter, which is essential for activation by enhancers such as the circadian clock genes BMAL1 and CLOCK. LPS-mediated NF-κB signalling interfered with the binding of these enhancers to the E-box, resulting in transcriptional repression. Conversely, treatment of macrophages with IL-4 and IL-13 led to an upregulation of MerTK protein expression, as demonstrated by RT-qPCR and western blotting. These findings suggest MerTK is a critical molecular switch in macrophage polarisation. Extending this investigation to MSCs-mediated effects, MSCs were generated from induced pluripotent stem cells (iPSCs), which offer a scalable and less invasive alternative to traditional MSCs sources limited by donor variability and invasive harvesting procedures. iMSCs were initially generated via directed differentiation of iPSCs in the presence of the transforming growth factor beta (TGFβ) inhibitor SB431542. The resulting iPSCs-derived MSCs (iMSCs) displayed a typical spindle-shaped morphology and expressed characteristic MSCs surface markers (CD73, CD90, CD105) with minimal CD45 expression, as confirmed by flow cytometry. Notably, the condition medium of iMSCs exhibited strong anti-inflammatory activity and upregulated MerTK expression, while exosomes isolated using Exo-spin™ mini-HD columns from iMSCs enhanced STAT3 phosphorylation, as evidenced by western blot analysis. These results suggested a paracrine mechanism through which MSCs promoted an anti-inflammatory macrophage phenotype. Collectively, these findings identified MerTK in macrophages as a key regulator of MSCs–macrophages crosstalk, modulated by both inflammatory signalling and circadian rhythms. They offer novel mechanistic insights into how iMSCs modulate the immune response and promote tissue regeneration through their interaction with macrophages, highlighting promising therapeutic potential for treating inflammatory disorders and supporting tissue repair.