Stem Cell Research & Therapy | Full text …

Abstract Introduction

Bone marrow mesenchymal stem cells (BMMSCs) are a heterogeneous population of postnatal precursor cells with the capacity of adhering to culture dishes generating colony-forming unit-fibroblasts (CFU-F). Here we identify a new subset of BMMSCs that fail to adhere to plastic culture dishes and remain in culture suspension (S-BMMSCs).

To catch S-BMMSCs, we used BMMSCs-produced extracellular cell matrix (ECM)-coated dishes. Isolated S-BMMSCs were analyzed by in vitro stem cell analysis approaches, including flow cytometry, inductive multiple differentiation, western blot and in vivo implantation to assess the bone regeneration ability of S-BMMSCs. Furthermore, we performed systemic S-BMMSCs transplantation to treat systemic lupus erythematosus (SLE)-like MRL/lpr mice.

S-BMMSCs are capable of adhering to ECM-coated dishes and showing mesenchymal stem cell characteristics with distinction from hematopoietic cells as evidenced by co-expression of CD73 or Oct-4 with CD34, forming a single colony cluster on ECM, and failure to differentiate into hematopoietic cell lineage. Moreover, we found that culture-expanded S-BMMSCs exhibited significantly increased immunomodulatory capacities in vitro and an efficacious treatment for SLE-like MRL/lpr mice by rebalancing regulatory T cells (Tregs) and T helper 17 cells (Th17) through high NO production.

These data suggest that it is feasible to improve immunotherapy by identifying a new subset BMMSCs.

Bone marrow mesenchymal stem cells (BMMSCs) are hierarchical postnatal stem/progenitor cells capable of self-renewing and differentiating into osteoblasts, chondrocytes, adipocytes, and neural cells [1,2]. BMMSCs express a unique surface molecule profile, including expression of STRO-1, CD29, CD73, CD90, CD105, CD146, Octamer-4 (Oct4), and stage-specific embryonic antigen-4 (SSEA4) [3,4]. It is generally believed that BMMSCs are negative for hematopoietic cell markers such as CD14 and CD34 [5-13]. BMMSCs have been widely used for tissue engineering [14-16]. Recently, a growing body of evidence has indicated that BMMSCs produce a variety of cytokines and display profound immunomodulatory properties [17-19], perhaps by inhibiting the proliferation and function of several major immune cells, such as natural killer cells, dendritic cells, and T and B lymphocytes [17-20]. These unique properties make BMMSCs of great interest for clinical applications in the treatment of different immune disorders [17,21-24].

BMMSCs are thought to be derived from the bone marrow stromal compartment, initially appearing as adherent, single colony clusters (colony-forming unit-fibroblasts [CFU-F]), and subsequently proliferating on culture dishes [25]. To date, the CFU-F assay has been considered one of the gold standards for determining the incidence of clonogenic BMMSC [26,27]. Since BMMSC are a heterogeneous population of stem cells, it is critical to identify whether BMMSC contain unique cell subsets with distinctive functions, analogous to the hematopoietic stem/progenitor cell system. In this study, we identified a subset of mouse BMMSCs in culture suspension and determined their immunomodulatory characteristics.

Female C3H/HeJ, C57BL/6J, and C3MRL-Faslpr/J mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). Female immunocompromised mice (Beige nude/nude XIDIII) were purchased from Harlan (Indianapolis, IN, USA). All animal experiments were performed under the institutionally approved protocols for the use of animal research (USC #10874 and 10941).

Anti Oct4, SSEA4, Runx2, OCN, active catenin and catenin were purchased from Millipore (Billerica, MA, USA). Anti alkaline phosphatase (ALP) antibody was purchased from Abcam (Cambridge, MA, USA). Anti Sca-1-PE, CD34-PE, CD34-FITC, CD45-PE, CD73-PE, CD4-PerCP, CD8-FITC, CD25-APC, CD3 and CD28 antibodies were purchased from BD Bioscience (San Jose, CA, USA). Anti Foxp3-PE, IL17-PE, and IFN-APC antibodies were purchased from eBioscience (San Diego, CA, USA). Unconjugated anti CD34, CD73, and CD105, NOS2 were purchased from Santa Cruz Biosciences (Santa Cruz, CA, USA). Anti actin antibody was purchased from Sigma (St. Louis, MO, USA).

The single suspension of bone marrow derived all nucleated cells (ANCs) from femurs and tibias were seeded at a density of 15 106 into 100 mm culture dishes (Corning, NY, USA) at 37C and 5% CO2. Non-adherent cells were removed after two days and attached cells were maintained for 16 days in alpha minimum essential medium (-MEM, Invitrogen, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (FBS, Equitech-bio, Kerrville, TX, USA), 2 mM L-glutamine, 55 M 2-mercaptoethanol, 100 U/ml penicillin, and 100 g/ml streptomycin (Invitrogen). Colony-forming attached cells were passed once for further experimental use.

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