Long-term expansion and enhanced osteogenic potential of Macaca MSCs via BMP signaling modulation

Highlights

• BMP2, in combination with FGF2, not only enhanced the proliferation of bone marrow-derived MSCs but also strengthened their osteogenic potential after short-term expansion in vitro.

• Both BMP and FGF signals are necessary and sufficient for the long-term expansion of MSCs with high osteogenic potential.

Abstract

Mesenchymal stem cells (MSCs) are a potential source of osteoblasts for the treatment of osteoporosis, but how to better preserve the stemness of MSCs in vitro culture conditions is the main challenge for MSC transplantation. The use of fibroblast growth factor 2 (FGF2) supplement has been described and used extensively to increase the expansion of MSCs. Cumulative evidence indicates that bone morphogenetic protein 2 (BMP2; a member of the TGF-β superfamily) is a secreted protein that promotes bone formation, which can regulate cell growth, differentiation, and development. Here we found that BMP2, in combination with FGF2, not only enhanced the proliferation of Macaca bone marrow-derived MSCs but also strengthened their osteogenic potential after short-term expansion in vitro. During long-term expansion, these cells still retained their osteogenic potential as well as other functional characteristics of pluripotent MSCs, which are gradually lost in the absence of BMP2. In addition, the BMP antagonist Noggin did not affect MSC expansion and the osteogenic potential. This study demonstrates that the regulation of BMP signaling can maintain the effectiveness of MSCs during expansion, which promotes the clinical application of MSCs in bone repair.

Introduction

Osteoporosis is a disabling bone disease characterized by reduced bone mineral density and increased risk of fractures. Approximately 200 million individuals worldwide suffer from osteoporosis (Cooper, 1999). Many patients with osteoporosis do not receive appropriate treatment due to concerns about drug safety. Currently, cell-based therapies have emerged as promising treatment options. Among them, mesenchymal stem cells, with their self-renewal capacity and multilineage differentiation potential, are a promising source for osteogenic tissue engineering (Frieedenstein et al., 1968; Nombela-Arrieta et al., 2011). MSCs can be obtained from a wide variety of sources, including bone marrow, adipose tissue, and umbilical cord. To date, bone marrow-derived MSCs and fat-derived MSCs have been most accurately described (Cislo-Pakuluk and Marycz, 2017; Marycz et al., 2015). Bone marrow-derived MSCs are more stable both in standard culture and in osteogenic culture (Marycz et al., 2015). The use of bone marrow-derived MSCs in cell-based tissue engineering therapy is proposed as a suitable alternative because the heterogeneity of the population reflects the special potential of differentiation into the bone and bone-related cells (Neuhuber et al., 2008).

In clinical trials, bone marrow-derived MSCs have been the most commonly used source of MSCs (Ankrum and Karp, 2010), while these cells are a rare population that constitutes only approximately 0.001 %–0.01 % of the total bone marrow mononuclear cells (Pittenger et al., 1999). Therefore, MSCs isolated for clinical applications usually require extensive in vitro expansion before their therapeutic use. However, these cells have limited cell proliferation capacity, and gradually lose their stem cell properties during expansion in vitro, leading to reduced therapeutic potential (Banfi et al., 2000; Bonab et al., 2006; Wang et al., 2016). Long-term expansion of MSCs in vitro showed great impairment of differentiation potential, such as complete loss of osteogenic potential and reduced adipogenic potential (Geissler et al., 2012). There is an urgent need to find a way to improve it. FGF2 can improve the expansion of MSCs (Bianchi et al., 2003; Quarto et al., 2001; Tsutsumi et al., 2001), but FGF2 does not prevent the progressive loss of cell differentiation capacity. Therefore, the main challenge is to determine the factors that support MSC amplification while maintaining their osteogenic capacity.

To address the factors, we noticed the process of bone formation during embryonic development. BMP signals regulate bone growth and bone homeostasis. The absence of BMP signaling is sufficient to cause severe skeletal growth retardation or bone loss in mice (Tan et al., 2007; Zhang et al., 2005). Reducing BMP signaling by removing BMP receptors impairs osteoblast differentiation and maturation (Yoon et al., 2005). The combination of BMP and FGF proteins improves the survival of MSCs (Hahn et al., 2008). Moreover, the combination of BMP and FGF can inhibit the induction of BMP in the rat femoral defect model (Wang et al., 2013). However, whether the combination of BMP2 and FGF2 supports MSC expansion and maintains their osteogenic differentiation potential is unclear.

In this context, we designed a series of cellular and biochemical experiments to investigate the role of the combination of BMP2 and FGF2 in the expansion of Macaca bone marrow-derived MSCs. Our data reveal that during multiple passages, the combination of BMP2 and FGF2 supports the extensive expansion of Macaca bone marrow-derived MSCs, while maintaining strong osteogenic potential. Furthermore, our results indicate that inhibition of endogenous BMP signals does not affect MSC expansion and osteogenic potential.