Heterogeneity of the biological properties and gene expression profiles of murine bone marrow stromal cells

https://doi.org/10.1016/j.biocel.2013.07.015Get rights and content

Abstract

Although mesenchymal stromal cells (MSCs) have demonstrated great therapeutic potential, the heterogeneity of MSCs may be responsible for the incongruent data obtained in MSC-based preclinical studies and clinical trials. Here, four mouse clonal MSC lines, termed MSC1, MSC2, MSC3, and MSC4, were isolated and extensively characterized. MSC4 cells grew most rapidly and formed colonies of the largest size, whereas MSC3 cells exhibited the slowest growth and formed only a few tiny clusters. MSC4 cells could differentiate into adipocytes, osteoblasts, and chondrocytes in vitro, and more importantly, establish hematopoietic microenvironment in vivo; whereas the other lines displayed uni-adipogenic, osteo-chondrogenic, or non-differentiation potential. All lines were positive for Sca-1, CD106, and CD44; MSC4 was also positive for CD90.2. In terms of immunosuppressive capacity, MSC2, MSC3, and MSC4 cells exerted clear inhibitory effects on lymphocyte proliferation, whereas MSC1 did not. Further investigation revealed that the NO and not the PGE2 pathway may play a role in the different immunomodulatory effects of the cell lines. To clarify the molecular basis of this heterogeneity, we employed RNA sequencing to compare the gene expression profiles of the four subtypes, revealing a relationship between gene expression and variability in subtype function. This study provides novel information about the heterogeneity of MSCs and insight into the selection of optimal cell sources for therapeutic applications.

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.

References (46)

  • A. Augello et al.

    Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis

    Arthritis & Rheumatism

    (2007)
  • S. Bae et al.

    Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts

    Cell and Tissue Research

    (2009)
  • S. Bae et al.

    Combined omics analysis identifies transmembrane 4 L6 family member 1 as a surface protein marker specific to human mesenchymal stem cells

    Stem Cells and Development

    (2011)
  • E.J. Bassi et al.

    Exploring the role of soluble factors associated with immune regulatory properties of mesenchymal stem cells

    Stem Cell Reviews

    (2012)
  • B.A. Bunnell et al.

    New concepts on the immune modulation mediated by mesenchymal stem cells

    Stem Cell Research & Therapy

    (2010)
  • P.F. Caimi et al.

    Emerging therapeutic approaches for multipotentmesenchymal stromal cells

    Current Opinion in Hematology

    (2010)
  • L.M. Calvi et al.

    Osteoblastic cells regulate the haematopoietic stem cell niche

    Nature

    (2003)
  • G. Chamberlain et al.

    Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing

    Stem Cells

    (2007)
  • E. Charytonowicz et al.

    PPAR (agonists enhance ET-743-induced adipogenic differentiation in a transgenic mouse model of myxoid round cell liposarcoma

    Journal of Clinical Investigation

    (2012)
  • J. Chen et al.

    ToppGene Suite for gene list enrichment analysis and candidate gene prioritization

    Nucleic Acids Research (Web Server issue)

    (2009)
  • B.M. Choi et al.

    Nitric oxide as a pro-apoptotic as well as anti-apoptotic modulator

    Journal of Biochemistry and Molecular Biology

    (2002)
  • M.B. Eslaminejad et al.

    Murine mesenchymal stem cell isolated and expanded in low and high density culture system: surface antigen expression and osteogenic culture mineralization

    In Vitro Cellular & Developmental Biology: Animal

    (2009)
  • S. Ghannam et al.

    Immunosuppression by mesenchymal stem cells: mechanisms and clinical applications

    Stem Cell Research & Therapy

    (2010)
  • Cited by (32)

    • Strategies to address mesenchymal stem/stromal cell heterogeneity in immunomodulatory profiles to improve cell-based therapies

      2021, Acta Biomaterialia
      Citation Excerpt :

      Use of cMSCs enables the rational selection and expansion of therapeutically relevant homogenous MSC sources from each donor to eliminate MSC variability between donors, expansion, and banking protocols and the resulting MSC end product. These and follow-on studies have since provided substantial evidence that donor whole MSC populations comprise mixtures of distinct MSC clonal subsets [69] with high and low differentiation potential (i.e., tripotent vs. unipotent) [67,74,75], distinct morphological phenotypes, differing proliferative capacities [93], and distinct immunomodulatory/regenerative potentials [75,78]. This MSC intra-population heterogeneity lies at the intersection of intrinsic cell biological and extrinsically introduced banking/processing heterogeneity.

    • Mesenchymal stem cell therapies in brain disease

      2019, Seminars in Cell and Developmental Biology
    • Mesenchymal stromal cells mitigate experimental colitis via insulin-like growth factor binding protein 7-mediated immunosuppression

      2016, Molecular Therapy
      Citation Excerpt :

      All of the animal procedures were reviewed and approved by the Sun Yat-Sen University Institutional Animal Care and Use Committee. Mouse MSCs were isolated from the bone marrow of 8-week-old C57BL/6 mice according to the improved low-density culture method reported previously.19,47 In brief, femurs and tibiae were removed and placed on ice in 5 ml L-Dulbecco's modified essential medium (DMEM) complete medium.

    • Nestin<sup>+</sup> kidney resident mesenchymal stem cells for the treatment of acute kidney ischemia injury

      2015, Biomaterials
      Citation Excerpt :

      For colony forming unit-fibroblast assay (CFU-F), 5000 Nestin+ cells were plated on tissue culture plastic and cultured in above-mentioned cell growth medium. After 10 days of cultivation, the number of colonies containing greater than 50 cells were assessed by inverted microscopy using an Olympus IX71 and quantified with the aid of a DP manager program (Olympus, Japan) [22]. Nestin+ cells proliferation was evaluated with Click-iT® EdU cell Fluor Cell Proliferation Assay Kit (Invitrogen, USA).

    View all citing articles on Scopus
    View full text