Supportive angiogenic and osteogenic differentiation of mesenchymal stromal cells and endothelial cells in monolayer and co-cultures

Abstract

Sites of implantation with compromised biology may be unable to achieve the required level of angiogenic and osteogenic regeneration. The specific function and contribution of different cell types to the formation of prevascularized, osteogenic networks in co-culture remains unclear. To determine how bone marrow-derived mesenchymal stromal cells (BMSCs) and endothelial cells (ECs) contribute to cellular proangiogenic differentiation, we analysed the differentiation of BMSCs and ECs in standardized monolayer, Transwell and co-cultures. BMSCs were derived from the iliac bone marrow of five patients, characterized and differentiated in standardized monolayers, permeable Transwells and co-cultures with human umbilical vein ECs (HUVECs). The expression levels of CD31, von Willebrand factor, osteonectin (ON) and Runx2 were assessed by quantitative reverse transcriptase polymerase chain reaction. The protein expression of alkaline phosphatase, ON and CD31 was demonstrated via histochemical and immunofluorescence analysis. The results showed that BMSCs and HUVECs were able to retain their lineage-specific osteogenic and angiogenic differentiation in direct and indirect co-cultures. In addition, BMSCs demonstrated a supportive expression of angiogenic function in co-culture, while HUVEC was able to improve the expression of osteogenic marker molecules in BMSCs.

Approaches to mimic the complexity of the skeletal mesenchymal stem/stromal cell niche in vitro

“…The signature and proliferation capacity of MSCs is also compromised in ECM-related disorders, such as tissue fibrosis -where an excessive deposition of ECM is observed (Usunier et al, 2014)or osteoarthritis (Maldonado and Nam, 2013) -which is linked to a resilient ECM degradation mediated primarily by MMPs and to an over-activation of osteoclast activity (Maldonado and Nam, 2013). Disruption of such control mechanisms generates aberrant ECM, both structurally and mechanically altered, leading to abnormal behaviors of cells residing in the niche and, ultimately, to enormous repercussions on the overall tissue homeostasis and functionality (Bonnans et al, 2014;Cox and Erler, 2011). BM niches can also be targeted by metastasizing cancer cells (discussed in more detail below), where a malignant vicious cycle between niche and tumor cells is created, adapting the ECM dynamics to each step of tumor progression (Herrmann et al, 2019;Lu et al, 2012).…”

“…Stem cell behavior, tissue formation and regeneration as well as survival of bone grafts are under the control of the blood vessels, which supply oxygen and nutrients to the cells (Böhrnsen and Schliephake, 2016;. Controlled diffusion of ROS, BM blood-vessel-forming ECs and vascular integrity determine and regulate HSPC as well as MSC localization and functionality (Fehrer et al, 2007;Gomariz et al, 2018;Langen et al, 2014;.…”

“…MSCs attenuate activation, proliferation and angiogenesis of ECs, through the production of MMP-1 (Zanotti et al, 2016) and ROS, leading to EC apoptosis, capillary degeneration (Marfy-Smith and Clarkin, 2017; Otsu et al, 2009), and, finally, disease (Cipriani et al, 2007). In contrast, MSC-EC crosstalk stimulates proliferation and osteogenesis in MSCs and angiogenesis in ECs (Bidarra et al, 2011;Böhrnsen and Schliephake, 2016). While BM endothelial progenitors, considered to be CD34 + or CD133 + cells, downregulate osteogenesis in MSCs (Duttenhoefer et al, 2015), EC progenitorderived growth factors are of critical importance for MSC engraftment, stemness, and repopulation in secondary grafts and osteogenesis (Lin et al, 2014).…”

Design of biomimetic cellular scaffolds for co-culture system and their application

“…Indirect co-culture of bone marrow–derived mesenchymal stromal cells and human umbilical vein ECs (HUVECs) on the transwell system yields an optimally organized vascular structure. 48 Angiogenic growth factors, such as angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor (VEGF) accelerate proliferation and promote intercellular tight junction formation in ECs, as well as enhance angiogenesis.…”

Magnetocontrolled protein membranes for cell cultures co-cultivation

“…Growth factors secreted by mesenchymal stem cells control in situ endothelial cells, fibroblasts, and chondrocytes 30 . Indirect co-cultivation of bone marrow-derived mesenchymal stromal cells and human umbilical vein endothelial cells in the "transwell system" leads to the formation of an optimally organized vascular structure 31 . A system for co-culturing C2C12 and 3T3-L1 cells was created to understand the formation of muscle and fat cells in animals, which can be used to model muscle degeneration, apoptosis, and muscle regeneration 32 .…”