The International Journal of Biochemistry & Cell Biology
Heterogeneity of the biological properties and gene expression profiles of murine bone marrow stromal cells
Introduction
Mesenchymal stromal cells (MSCs) have been isolated from various adult tissues and are defined as adherent fibroblast-like cells that can differentiate into multiple types of mesodermal cells (e.g., osteoblasts, adipocytes, and chondrocytes) (Bianco et al., 2008, Chamberlain et al., 2007, Uccelli et al., 2008). Human MSCs are considered to be therapeutically valuable for tissue repair and treatment of immune system-mediated disease because of their potential for differentiation into diverse lineages, their broad immunoregulatory capability and paracrine effects, and their ability to home to injured sites (Chamberlain et al., 2007, Uccelli et al., 2008, Shi et al., 2010, Ghannam et al., 2010, Wang et al., 2011, Ma, 2010, Caimi et al., 2010). Although preclinical studies and early-stage clinical trials have yielded promising results in various disease models, conflicting results have been reported. These between-study differences may be due to variations in the methods used for cell harvesting and expansion, the donor variance use of different tissue sources of MSCs, the lack of treatment schedule standardization, and (more importantly) the heterogeneity among MSCs obtained in different laboratories (Pevsner-Fischer et al., 2011, Phinney, 2012). Because there are no defined molecular markers of MSCs, MSCs are usually isolated via adhesion to plastic and further characterized by a panel of cell surface markers and multilineage differentiation abilities, yielding a heterogeneous subset ensemble of progenitors and lineage-committed cells. It has been suggested that the use of mixed cell populations explains some of the contradictory clinical results (Rosenzweig, 2006, Galipeau, 2013). Thus further characterization of MSC subpopulations is essential to obtain consistent clinical results.
To date, several studies have demonstrated the heterogeneity of MSC. Clonal analysis of human bone marrow MSC revealed heterogeneity in terms of osteogenic differentiation potential and the formation of bone marrow microenvironments in vivo (Kuznetsov et al., 1997, Okamoto et al., 2002). Clonal heterogeneity in the osteogenic capacity of murine bone marrow MSC both in vitro and in vivo has also been described (Satomura et al., 2000). In addition, proliferative potential also differs among MSCs. Human bone marrow MSCs have been shown to be a heterogeneous population of progenitor and lineage-committed cells featuring a broad range of proliferation potential and differentiation potency (Russell et al., 2011). However, the phenotypes of heterogeneous bone marrow MSC remain poorly described, and a little information about the genes responsible for the cellular functions of bone marrow MSC is available (Bae et al., 2009, Bae et al., 2011, Götherström et al., 2005). Specifically, few comparative analyses of immunomodulatory capacity among different bone marrow MSC subsets have been performed, even though such cells are often used to treat immune system disorders (Uccelli et al., 2008, Shi et al., 2010, Ghannam et al., 2010).
The isolation and expansion of murine bone marrow MSC have proven to be much more difficult than that of their counterparts from other species because of the low incidence of MSCs in murine bone marrow and the unwanted growth of contaminated hematopoietic cells (Sun et al., 2003). So information about murine bone marrow MSC heterogeneity is even more lacking. Thus, it is urgently needed to clarify the characteristics of subpopulations within mouse bone marrow MSCs, including their functional attributes and the molecular basis of their heterogeneity.
In this study, we obtained four distinct subtypes of bone marrow MSCs using low-density culture with clonal selection and compared the biological characteristics of these lines in terms of growth characteristics, differentiation potential, phenotype, gene expression, and immunomodulatory capability. The resulting data provide important quantitative and qualitative insights into the identification of optimal cell sources for therapeutic applications.
Section snippets
Isolation and expansion of mouse bone marrow MSC subtypes
MSCs were isolated from murine femoral bone marrow by an improved low density culture method (Eslaminejad and Nadri, 2009, Meirelles and Nardi, 2003, Peister et al., 2004). In brief, the medullary canal was flushed with l-Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY) containing 10% (v/v) fetal calf serum (FCS; Hyclone, Logan, UT) and filtered through a 70-μm pore-sized steel mesh to collect bone marrow cells. After the red blood cells were lysed, the bone marrow cells were
Establishment of 4 mouse bone marrow MSC subtypes, and investigation of morphology, growth characteristics, and CFU-F capability
Through passaging at low density in combination with clonal selection, 4 distinct plastic-adherent cell lines were established (MSC1, MSC2, MSC3, and MSC4). These cell lines differed in terms of morphology: MSC1 cells were small and oval, with a clear nucleus and nucleolus, whereas MSC2 cells had a fibroblast-like morphology, with branched projections. MSC3 cells were fusiform, and MSC4 cells grew as small polygons (Fig. 1A). Among the 4 types of MSCs, MSC4 cells grew most rapidly, at almost
Discussion
In routine mesenchymal cell culture, bone marrow MSCs are usually heterogeneous populations containing multipotent MSCs and committed lineages (Kuznetsov et al., 1997, Okamoto et al., 2002, Satomura et al., 2000, Russell et al., 2011). Thus the use of these mixed cell populations may at least partially explain the variations in research or preclinical applications developed by different laboratories (Rosenzweig, 2006, Galipeau, 2013). In the present study, we obtained four mouse MSC subtypes
Financial support
This work was funded by National Basic Research Program of China (2012CBA01302, 2009CB522104 and 2010CB945401), National Natural Science Foundation of China (U0932006, 31171398, 81170367, and 81270646), Key Scientific and Technological Projects of Guangdong Province (2007A032100003), Key Scientific and Technological Program of Guangzhou City (2008A1-E4011-5 and 2010U1-E00551).
Conflict of interest
We declare that we have no conflict of interest.
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