Abstract Body (Do not enter title and authors here): Background and aim: Transplantation of mesenchymal stromal cells (MSCs) has been developed as a promising strategy for treating ischemic heart diseases. Transplanted MSCs will not survive long; most of them die by apoptosis, proposing the paracrine effect as a mechanism of their beneficial effect. Although transplantation of MSCs increases the number of M2-like reparative macrophages (Mφ) in the recipient ischemic tissues, the underlying mechanisms have not been fully elucidated. Recently, we found that MSCs express high levels of efferocytosis-related genes, especially relating to the bridge between apoptotic cells and Mφ (e.g., Mfg-e8, Gas6, Pros1, and Thbs1). On the other hand, it has been known that Mφ changes their polarity toward reparative M2-like by engulfing the apoptotic cells-mimicking liposomes. Thus, we hypothesized that the engulfment of apoptotic MSCs by Mφ may exert the therapeutic effect of MSCs transplantation. The aim was to examine whether the engulfment of apoptotic MSCs alters the phenotype of Mφ.
Methods: Commercially available monocyte cell line; THP-1, was differentiated into 4 subtypes of Mφ (M0, M1, M2a, and M2c). Human induced pluripotent stem cells-derived MSCs (iMSCs) were treated with the mixture of inhibitors for Bcl-2, BCL-XL, and MCL-1 for 20 min at 37 °C to induce apoptosis, and labeled with a fluorescent pH probe (pHrodo). THP-1-Mφ and apoptotic iMSC were cocultured overnight, and the engulfment ratio was examined by flow cytometry. The transcriptome of pHrodo-positive and pHrodo-negative-Mφ was compared comprehensively to elucidate whether the engulfment of apoptotic iMSC changes the transcriptome in each subtype of Mφ.
Results: Engulfment ratio was almost identical among all subtypes of Mφ (M0: 39.1±8.7% vs. M1: 32.1±10.3% vs. M2a: 40.1±14.5% vs. M2c: 43.8±11.4%). Confocal microscopic observation demonstrated that most of the engulfed iMSCs were localized in the lysosomes. RNA-Seq data revealed that M2 Mφ-related genes were upregulated by engulfing apoptotic iMSCs in M2a and M2c Mφ (Figure, filled arrows). Finally, the CD206 and CD163 mRNA expressions determined by real-time PCR were significantly increased in pHrodo-positive, iMSC-engulfing M2a and M2c Mφ, respectively (CD260; 1.54±0.60-fold, CD163; 1.58±0.36-fold).
Conclusion: Our results suggested that the engulfment of apoptotic MSCs by Mφ might contribute to the therapeutic effect of MSCs transplantation in addition to the secretion of cytokines/chemokines/exosomes.