Improving ex-vivo expansion of mesenchymal stromal/stem cells using acellular fetal membranes

Abstract

Mesenchymal stromal/stem cells (MSCs) have considerable potential in the fields of cell therapies, tissue engineering, and regenerative medicine. According to clinicaltrials.gov, MSCs are employed in more than 700 registered clinical trials as potential treatments. However, the clinical application of MSCs is limited by their low prevalence in the human body and inefficient methods for large-scale ex-vivo production. During ex-vivo expansion, MSCs experience a vastly different environment compared to their natural microenvironment (i.e. their niche), and these environmental differences are likely to be main drivers for the loss of key MSC properties. In this study, the general aim was to investigate the effect of two main components of the MSC niche on decidua-derived MSCs (DMSCs) from human placenta during ex-vivo expansion; the extracellular matrix (ECM) and extracellular vesicles (EVs).

Coatings produced from ECM are promising surfaces for the improved ex-vivo expansion of MSCs. However, identifying a readily available source of ECM to generate these coatings is the bottleneck of this technology. In Chapter 2 of this thesis, ECM coatings derived from decellularised fetal membranes were assessed as suitable substrates for MSC expansion. The fetal membrane’s two main components, the amnion and the chorion, were separated, decellularised and processed further to produce solubilised forms of the decellularised amniotic membrane (s-dAM) and decellularised chorionic membrane (s-dCM). DMSCs were more proliferative, smaller in size (a measure of MSC potency, and exhibited greater adopogenic and osteogenic differentiation capacity when cultured on s-dAM compared to controls. Additionally, long-term culture studies revealed that late passage DMSCs (passage 8) cultured on s-dAM had decreased cell diameter over three passages. These data support the use of s-dAM as a substrate for improved MSC expansion.

In addition to the ECM, extracellular vesicles are another important component of the MSC niche. However, the contribution of ECM and EVs has not been explored from an MSC expansion point of view. In Chapter 3, the effect of adding MSC-derived EVs to DMSCs cultured on the ECM coatings described in Chapter 2 was assessed. Addition of EVs to the DMSCs growing on Matrigel improved their attachment. However, regardless of the presence of EVs, DMSCs showed significantly better attachment on s-dAM. Furthermore, addition of EVs to DMSCs growing on s-dAM improved DMSC proliferation, migration and osteogenic capacity. The total antioxidant capacity of DMSCs growing on Matrigel, s-dAM and s-dCM increased, regardless of whether EVs were added to DMSCs or not. These data illustrate the relative contribution of ECM and EVs towards MSC expansion and show that supplementing the MSC culture with EVs can regulate certain MSC properties.

In addition to issues of large-scale ex-vivo expansion of MSCs, their clinical application is limited due to their low survival rates and poor engraftment after delivery. A number of factors contribute to these issues including cell damage due to shear stress during injection, leakage of cells from the injection site, and lack of appropriate interactions with surrounding cells and extracellular matrix. In Chapter 4 of this thesis, s-dAM and s-dCM were investigated for their potential to form 3D thermoreversible and injectable hydrogels to be used as a cell delivery carrier for MSCs. At 37ºC, both s-dAM and s-dCM formed gels at concentrations of 4 and 8 mg/mL. DMSCs growing on s-dAM showed improved proliferation, adipogenesis and osteogenesis compared to TCP. Both s-dAM and s-dCM had shear thinning properties, and were therefore injectable. DMSCs embedded in both s-dAM and s-dCM had a viability of ~60% after injection and showed improved proliferation compared to Matrigel. These data support that the ECM from both s-dAM and s-dCM can be processed to produce thermoreversible and injectable hydrogels, and s-dAM hydrogels promote key properties of DMSCs.