Expansion of human bone marrow-derived mesenchymal stromal cells with enhanced immunomodulatory properties

Abstract

Background: Mesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD.

Methods: Human bone marrow-derived MSCs were expanded at high-confluency (MSC_HC) and low-confluency state (MSC_LC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSC_HC or MSC_LC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45⁺ blood count and histopathological examination.

Results: As compared to MSC_LC, MSC_HC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSC_HC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSC_HC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSC_LC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSC_HC as well as enriched pathways including cytokine-cytokine receptor interactions, cell adhesion and PI3K-AKT signalling_. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSC_HC showed greater suppression of inflammation and improved outcomes compared to MSC_LC and saline controls.

Conclusion: Our study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.

Keywords: Cell culture; Confluency; Graft-versus-host disease; Immunomodulation; Mesenchymal stromal cells; T cells.