Mesenchymal stem cells (MSCs) are capable of self-replication and multilineage differentiation, making them a promising cell source for regenerative medicine owing to their low risk of tumorigenesis. MSCs derived from tissues such as the bone marrow (Haynesworth et al., 1992, Pittenger et al., 1999), placenta (In 't Anker et al., 2004), adipose tissue stromal vascular fraction (Halvorsen et al., 2000, Zuk et al., 2001), and umbilical cord (He et al., 2017, Romanov et al., 2003) exhibit similar expression profiles and biological functions as cell surface markers; however, their characteristics differ based on their source tissue. Human umbilical cord perivascular cells (HUCPVCs) are immature MSCs derived from fetal appendages such as Wharton’s jelly in the umbilical cord and the placental amniotic membrane (Sarugaser et al., 2009, Sarugaser et al., 2005, Wang et al., 2004). HUCPVCs harvested from human umbilical cord perivascular tissue constitute a population of MSCs and are extensively utilized as a cell source for various studies in MSCs (Gauthier-Fisher et al., 2020, Shohara et al., 2012, Szaraz et al., 2019, Szaraz et al., 2020, Zebardast et al., 2010). However, there is no currently known method to differentiate HUCPVCs into hard tissue-forming cells such as osteoblasts. In vitro studies using conditioned media from human bone marrow-derived mesenchymal stromal cell cultures demonstrated the osteogenic differentiation potential of HUCPVCs (Kajiyama et al., 2021, Kajiyama et al., 2015).

Transforming growth factor beta (TGF-β) is a cytokine that induces proliferation, differentiation, chemotaxis, and apoptosis in monocytic and epithelial cells, mesenchymal cells, and nerve cells (Kubiczkova et al., 2012). In mammals, TGF-β has three isoforms, namely TGF-β1, TGF-β2, and TGF-β3, each of which exhibit similar biological activities (Kingsley, 1994, Massague, 1992). Previous in vitro study using TGF-β1 by our group have shown that HUCPVCs differentiate into myofibroblast-like cells and lack the ability to form hard tissue when cultured in the presence of activated vitamin D3, a bone morphogenetic protein (BMP) inhibitor, and TGF-β1 (Nonoyama et al., 2021). A study of bone tissue engineering applications have shown that a combination of TGF-β, and growth factors showing higher proliferation ability with the active-form vitamin D enhance osteoblast differentiation (Bosetti et al., 2007). Therefore, if we can find clear factors or conditions under which HUCPVCs differentiate into osteoblast lineage cells, they could further contribute to autologous stem cell-based therapies, such as osteoarthritis and fracture treatments, and may be even more useful in treating people with these bone diseases.

Fibroblast growth factor 2 (FGF-2) is a cytokine that promotes angiogenesis and the proliferation of many cell types. In the Japanese dental field, a periodontal tissue regenerator with FGF-2 as an active ingredient was clinically introduced. In this formulation, FGF-2 is believed to trigger the initial process of periodontal tissue regeneration and regulate angiogenesis and extracellular matrix production by stimulating MSC proliferation in the periodontal ligament (Murakami et al., 1999, Murakami et al., 2003, Takayama et al., 2001). Additionally, FGF-2 has been shown to induce the proliferation of fibroblasts, vascular endothelial cells, and hard tissue-forming cells such as osteoblasts and osteocytes (Agas et al., 2013, Ikpegbu et al., 2018). Studies of osteogenesis and chondrogenesis have shown that the combined use of FGF-2 and other growth factors elicits additional effects (Song et al., 2017, Takahashi et al., 2005).

Here, we report the synergistic effect of FGF-2 and TGF-β1 on the differentiation of HUCPVCs into hard tissue-forming cells at the biochemical, cellular, morphological, genetic, and crystal engineering levels.