Characterization of bone marrow derived mesenchymal stem cells in suspension

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).

Methods: 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.

Results: 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.

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

Figure 1

Identification of suspension BMMSCs (S-BMMSCs). (A) Hypothetical model indicates that bone marrow all nucleated cells (ANCs) were seeded at 15 × 10⁶ into 100 mm culture dishes and incubated for two days at 37°C with 5% CO2, and subsequently non-attached cells from culture suspension were transplanted into immunocompromised mice subcutaneously using hydroxyapatite tricalcium phosphate (HA) as a carrier for eight weeks. Newly formed bone (B) by osteoblasts (arrow heads) and associated connective tissue (C) were detected in this non-attached cell transplants by H & E staining. Bar = 100 μm. (B) Hypothetical model of isolating S-BMMSCs. BMMSCs usually attach on culture dishes within two days; however, a small portion of BMMSCs in ANCs failed to attach to the dishes and remained in the suspension. The suspensions containing putative non-attached BMMSCs were collected and transferred to the extracellular matrix (ECM) coated dish with generating single colony clusters (CFU-F). These ECM-attached BMMSCs (S-BMMSCs) were sub-cultured on regular plastic culture dishes for additional experiments. (C) The number of plastic attached CFU-F from ANCs (1.5 × 10⁶ cells) is more than seven-fold higher than that derived from BMMSC-ECM adherent S-BMMSCs. (D) Proliferation rates of S-BMMSCs and BMMSCs were assessed by BrdU incorporation for 24 hours. The percentage of positive cells is significantly increased in S-BMMSCs when compared to BMMSCs. (E) S-BMMSCs exhibit a significant increase in population doublings when compared to BMMSCs. The results are representative of five independent experiments. Scale bars = 50 μm. ***P <0.001. The graph bar represents mean ± SD. BMMSCs, bone marrow mesenchymal stem cells; BrdU, bromodeoxyuridine; S-BMMSCs, BMMSCs in suspension; SD, standard deviation.

Figure 2

Multipotent differentiation of S-BMMSCs. (A) Alizarin Red S and alkaline phosphatase (ALP) staining showed that S-BMMSCs were similar to regular BMMSCs in osteogenic differentiation in vitro. (B) S-BMMSCs or regular BMMSCs (4 × 10⁶ cells/transplant) were transplanted into immunocompromised mice using HA/TCP (HA) as a carrier for eight weeks. Bone formation was detected in S-BMMSC and BMMSC transplants, evidenced by H & E staining. HA, hydroxyapatite tricalcium phosphate; B, bone; M, bone marrow; CT, connective tissue. Bar = 50 μm. (C-D) S-BMMSCs are capable of forming Oil Red O positive cells (C) and expression of pparγ2 and lpl mRNA as seen in regular BMMSCs (D). Glyceraldehyde 3-phosphate dehydrogenase (gapdh) was used as an internal control. The results are representative of five independent experiments. Scale bars = 100 μm. (E) Chondrogenic differentiation was assessed by Alcian blue staining for acidic sulfated mucosubstances, Pollak's Trichrome staining for collagen, and immunohistochemical staining for collagen type II. S-BMMSCs were able to differentiate into chondrocytes as observed in regular BMMSCs. Bar = 50 μm. The results are representative of three independent experiments. The graph bar represents mean ± SD. BMMSCs, bone marrow mesenchymal stem cells; S-BMMSCs, BMMSCs in suspension; SD, standard deviation.

Figure 3

S-BMMSCs express CD34. (A) Flow cytometric analysis showed that regular BMMSCs fail to express CD34, but are positive for CD45 antibody staining (21.4%). However, S-BMMSCs express both CD34 (23.4%) and CD45 (31.2%). (B) Flow cytometric analysis also showed that CD34⁺ S-BMMSCs were positive for anti CD73 (13.8%) and Oct4 (13.4%) antibody staining. IgG isotype staining groups were used as negative controls. (C, D) Western blot analysis indicated that S-BMMSCs express CD34 and mesenchymal surface molecules CD73 and CD105. In contrast, regular BMMSCs only express CD73 and CD105 (C). S-BMMSCs express CD34 at passage one to five (D). β-actin was used as a sample loading control. BMC, whole bone marrow ANC. (E) Immunocytostaining confirmed that S-BMMSCs are double positive for CD34/CD73 (triangle). Regular BMMSCs are negative for CD34 antibody staining and only positive for anti CD73 antibody staining. Bar = 100 μm. (F) Both BMMSCs and S-BMMSCs failed to differentiate into hematopoietic lineage under hematopoietic inductive conditions with EPO (upper panel) or without EPO (lower panel). Whole bone marrow cells and lineage negative cells were used as positive (yellow arrowheads) control. Bar = 100 μm. ANC, all nucleated cells; BMMSCs, bone marrow mesenchymal stem cells; EPO, erythropoietin; S-BMMSCs, BMMSCs in suspension.

Figure 4

S-BMMSCs showed superior therapeutic effect on SLE-like MRL/lpr mice. (A) Schema of BMMSC transplantation into MRL/lpr mice. (B) S-BMMSC and BMMSC treatment recover basal membrane disorder and mesangium cell over-growth in glomerular (G) (H&E staining). (C) S-BMMSC and BMMSC transplantation could reduce urine protein levels at two weeks post transplantation compared to the MRL/lpr group. S-BMMSCs offered a more significant reduction compared to BMMSCs. (D, E) The serum levels of anti-dsDNA IgG and IgM antibodies were significantly increased in MRL/lpr mice compared to controls (C3H). S-BMMSC and BMMSC treatments could reduce antibody levels but S-BMMSCs showed a superior treatment effect than BMMSC in reducing anti-dsDNA IgG antibody (D). (F) S-BMMSC and BMMSC treatments could reduce increased levels of anti nuclear antibody (ANA) in MRL/lpr mice. S-BMMSC showed a better effect in ANA reduction compared to BMMSC. (G) S-BMMSC and BMMSC treatments could increase the albumin level in MRL/lpr mice, which was decreased in controls. S-BMMSC treatments were more effective in elevating the albumin level compared to BMMSC treatment. (H) Flow cytometric analysis showed a reduced number of Tregs in MRL/lpr peripheral blood compared to control. BMMSC and S-BMMSC treatments elevated the number of Tregs. S-BMMSCs induced a more significant elevation of the Tregs level than BMMSCs. (I) Flow cytometric analysis showed an increased number of Th17 in MRL/lpr mice peripheral blood compared to control. Th17 were markedly decreased in BMMSC and S-BMMSC treated groups. S-BMMSC treatment induced a more significant reduction of Th17 cells than treatment with BMMSCs. *P <0.05; ** P <0.01; ***P <0.001. The graph bar represents mean ± SD. BMMSCs, bone marrow mesenchymal stem cells; Ig, immunoglobulin; S-BMMSCs, BMMSCs in suspension; SD, standard deviation; SLE, systemic lupus erythematosus; Tregs, regulatory T cells.

Figure 5

S-BMMSCs show up-regulated immunomodulatory properties through nitric oxide (NO) production. (A) NO levels in the supernatant of S-BMMSC and BMMSC culture were significantly higher in the INF-γ/IL-1β treated S-BMMSC group than in BMMSCs. (B-C) S-BMMSCs showed a significant reduction in the cell viability of activated SP cells compared to the cells cultured without BMMSCs (SP cell) and with BMMSCs (B). Both BMMSCs and S-BMMSCs showed a significantly increased rate of SP cell apoptosis compared to the SP cell only group but S-BMMSCs could induce higher SP cell apoptosis (C). (D-E) The induction of SP cell apoptosis by BMMSCs or S-BMMSCs was abolished in general NOS inhibitor L-NMMA-treated (D) and iNOS specific inhibitor 1400 W-treated (E) group. (F-H) Activated CD4⁺CD25^- T-cells and S-BMMSCs or BMMSCs were co-cultured in the presence of TGFβ1 and IL-2 with or without NOS inhibitor for three days. The floating cells were stained for CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs). Both BMMSCs and S-BMMSC up-regulated Tregs but S-BMMSCs showed a significant effect in up-regulating Tregs. (F). Interestingly, L-NMMA and 1400 W treatments resulted in an abolishing of S-BMMSC-induced up-regulation of Tregs (G, H). (I) BMMSCs and S-BMMSCs could inhibit Th17 differentiation in vitro. S-BMMSC could inhibit it more effectively. (J, K) L-NMMA (J) or 1400 W (K) could abolish the inhibition of Th17 differentiation by BMMSCs or S-BMMSCs. The results are representative of at least three independent experiments. *P <0.05; **P <0.01; ***P <0.001. The graph bar represents mean ± SD. BMMSCs, bone marrow mesenchymal stem cells; iNOS, inducible nitric oxide synthase; L-NMMA, L-NG-monomethyl-arginine; NOS, nitric oxide synthase; S-BMMSCs, BMMSCs in suspension; SD, standard deviation; SP, spleen; Tregs, regulatory T cells.