Abstract
This study was aimed to investigate the effects of cGMP xeno-/serum-free medium (XSF, Irvine Scientific) on the properties of human dental pulp stem cells (DPSCs). DPSCs, from passage 2, were cultured in XSF or fetal bovine serum (FBS)-supplemented medium, and sub-cultured up to passage 8. Cumulative population doublings (PDs) and the number of colony-forming-units (CFUs) were determined. qRT-PCR, ELISA, and in vitro assays were used to assess angiogenic capacity. Flow cytometry was used to measure CD73, CD90, and CD105 expression. Differentiation into osteo-, adipo-, and chondrogenic cell lineages was performed. DPSCs showed more elongated morphology, a reduced rate of proliferation at later passages, and lower CFU counts in XSF compared with FBS. Expression of angiogenic factors at the gene and protein levels varied in the two media and with passage number, but cells grown in XSF had more in vitro angiogenic activity. The majority of early and late passage DPSCs cultured in XSF expressed CD73 and CD90. In contrast, the percentage of CD105 positive DPSCs in XSF medium was significantly lower with increased passage whereas the majority of cells cultured in FBS were CD105 positive. Switching XSF-cultured DPSCs to medium supplemented with human serum restored the expression of CD105. The tri-lineage differentiation of DPSCs cultured under XSF and FBS conditions was similar. We showed that despite reduced CD105 expression levels, DPSCs expanded in XSF medium maintained a functional MSC phenotype. Furthermore, restoration of CD105 expression is likely to occur upon in vivo transplantation, when cells are exposed to human serum.
Highlights
IntroductionMesenchymal stromal cells (MSCs) isolated from human dental pulp (DPSCs) have been shown to have multipotent differentiation capacity into various cell lineages, such as, neural ectodermal cells (Gronthos et al 2002; Iohara et al 2006), osteoblasts (Jo et al 2007; Mori et al 2011; Mortada and Mortada 2018; Otaki et al 2007; Pettersson et al 2017; Tabatabaei and Torshabi 2017), adipocytes (Gronthos et al 2002; Iohara et al 2006; Jo et al 2007; Pettersson et al.2017), odontoblasts (Gronthos et al 2002), chondrocytes (Iohara et al 2006), and myoblasts (Zhang et al 2006)
Our study showed an increased secretion of vascular endothelial growth factor A (VEGFA) from dental pulp stem cells (DPSCs) cultured in xeno-/serum-free medium (XSF) medium at passages 2 and 6, and this was further confirmed by the evaluation of an in vitro angiogenesis assay, which showed enhanced capillary tube-like formation in XSF-cultured conditioned medium compared with FCS-cultured conditioned medium
This study demonstrated that current good manufacturing practice (cGMP)-defined xeno-free/serum-free medium could be used to expand DPSCs in vitro, and these cells maintain their ability for trilineage differentiation even though low numbers of the cells expressed the multipotency marker, CD105
Summary
Mesenchymal stromal cells (MSCs) isolated from human dental pulp (DPSCs) have been shown to have multipotent differentiation capacity into various cell lineages, such as, neural ectodermal cells (Gronthos et al 2002; Iohara et al 2006), osteoblasts (Jo et al 2007; Mori et al 2011; Mortada and Mortada 2018; Otaki et al 2007; Pettersson et al 2017; Tabatabaei and Torshabi 2017), adipocytes (Gronthos et al 2002; Iohara et al 2006; Jo et al 2007; Pettersson et al.2017), odontoblasts (Gronthos et al 2002), chondrocytes (Iohara et al 2006), and myoblasts (Zhang et al 2006). 2009) and exhibit odontogenic features when transplanted into immunocompromised mice (Gronthos et al 2002; Otaki et al 2007) They can differentiate into osteoblasts, producing living autologous fibrous bone tissue which forms lamellar bone-containing osteocytes after transplantation into immnunocomprised mice (Laino et al 2005). Various xeno-/serumfree medium formulations have been developed for in vitro culture of MSCs prior to cell transplantation (Bonnamain et al 2013; Fujii et al 2018; Hirata et al 2010; Khanna-Jain et al 2012; Mochizuki and Nakahara 2018; Xiao et al 2018). There are conflicting conclusions regarding the effect on cell proliferation in comparison with FBS-supplemented media (Fujii et al 2018; Hirata et al 2010; Khanna-Jain et al 2012; Xiao et al 2018)
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