Introduction and objectives Mesenchymal stem cells (MSC) have well-established anti-inflammatory properties and could potentially be used therapeutically in Acute Respiratory Distress Syndrome (ARDS).¹ MSC conditioned medium (MSC-CM) has been found to reproduce the beneficial effects of MSCs.² However, the impact of expansion on MSC secretome remains elusive. In the present study we assessed the expression of four potent MSC paracrine factors after prolonged in vitro culture, while investigating the effects of passaging on the in vitro properties of MSCs.

Methods Human Bone marrow-derived MSCs were expanded in vitro in αMEM with 16% FBS. Conditioned medium was collected from each passage and stored at −40°C. Scratch assays were undertaken using A549 cells treated with MSC-CM and control media. Proliferation of A549 cells was assessed via BrdU assay. Gene expression of expanded MSCs was assessed by RT-qPCR. VEGF and Angiopoietin in the MSC-CM were quantified by ELISA.

Results RT-qPCR revealed that MSCs express the anti-inflammatory genes PTGES2, FGF7 and ANGPT1. The expression of these genes doubled after 8 days in culture and subsequently decreased. (P < 0.0005, one way Anova, n = 2). The secretion of Angiopoietin and VEGF decreased with prolonged expansion (Figure 1). MSC-CM obtained after 8 days of culture induced more efficient wound healing compared to MSC-CM obtained following prolonged expansion (P < 0.005, one way Anova, n = 2). However, MSC-CM failed to affect the proliferation of A549 cells.

Conclusion We conclude that MSCs of early passages are more potent angiogenic inducers, while promoting wound healing in vitro. Thus, we provide important insights into the changes occurring after prolonged in vitro expansion, underlining the importance of using low passage MSCs in clinical trials for ARDS. In agreement with published data, we also found that MSCs do not induce cellular proliferation in the absence of stimulation.

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Abstract S11 Figure 1

Levels of secreted VEGF decrease after prolonged in vitro culture (*P<0.05, ***P<0.0005, n = 2)

References

1. Antunes MA, et al. Mesenchymal stem cell trials for pulmonary diseases. J Cell Biochem 2014;115:1023–1032.

2. Kordelas L, et al. MSC-derived exosomes: a novel tool to treat therapy-refractory graft-versus-host disease. LeukaemiaLeukemia 2014;28:970–973.