A novel method to isolate mesenchymal stem cells from mouse umbilical cord

Abstract

Mesenchymal stem cells (MSCs), derived from various tissues, are considered an ideal cell source for clinical use, among which MSCs from the umbilical cord exhibit advantages over those from adult tissues. In preclinical studies, mouse models and xenogeneic MSC treatment are most commonly used to imitate diseases and clinical practice, respectively. However, the efficiency of cross‑species therapy remains controversial, making it difficult to elucidate the underlying mechanisms. Thus, allogeneic therapy may be more instructive and meaningful in clinical use. To confirm this hypothesis, the present study established a novel method for the isolation and expansion of MSCs from mouse umbilical cords (mUC‑MSCs) to support in vivo experiments in mice. MSCs were isolated from mUCs and mouse bone marrow (mBM), and then identified by flow cytometry. The differences in mUC‑MSCs and mBM‑MSCs were analyzed using a growth curve and their differentiation ability. The results showed that the harvested cells exhibited general characteristics of MSCs and possessed the capacity for long‑term culture. Despite having similar morphology and surface antigens to MSCs derived from mouse bone marrow, the mUC‑MSCs showed differences in purification, proliferation, stem cell markers and differentiation. In addition to detailed characterization, the present study verified the presence of Toll‑like receptor 3 (TLR3), an important component of immune responses, in mUC‑MSCs. It was found that the activation of TLR3 upregulated the levels of stemness‑related proteins, and enhanced the secretion and mRNA levels of inflammatory cytokines in the pre‑treated mUC‑MSCs. Collectively, the results of the present study provide further insight into the features of newly established mUC‑MSCs, providing novel evidence for the selection of murine MSCs and their responses to TLR3 priming.

Figure 1.

Morphology, growth, surface antigens and cell cycle of MSCs derived from mUC-MSCs and mBM-MSCs. (A) Appearance of mUC-MSCs and mBM-MSCs at passages (a) 0, (b) 10 and (c) 17 (magnification, ×40; scale bar, 100 µm. (B) Growth curves of mUC-MSCs and mBM-MSCs. (C) Flow cytometric analysis of surface markers CD29, CD44, CD34, CD45 and CD11b in mUC-MSCs and mBM-MSCs. (D) DNA contents of mUC-MSCs and mBM-MSCs. mUC, mouse umbilical cord; mBM, mouse bone marrow; MSCs, mesenchymal stem cells; CD, cluster of differentiation.

Figure 2.

Stemness-related proteins in mUC-MSCs and mBM-MSCs. Western blot assay for the expression of Sox2, Nanog, Oct4 and Sall4 in (A) mUC-MSCs during long-term culture and (B) mBM-MSCs. mUC, mouse umbilical cord; mBM, mouse bone marrow; MSCs, mesenchymal stem cells; Sox2, Sex determining region Y-box 2; Oct4, octamer-binding transcription factor 4; Sall4, Spalt-like transcription factor 4.

Figure 3.

Adipogenic and osteogenic differentiation of mUC-MSCs and mBM-MSCs. (A) mUC-MSCs and mBM-MSCs were cultured in adipogenic induction medium for two cycles, followed by (a) Oil red O staining and (b) RT-qPCR analysis of mRNA levels of adiponectin. (B) mUC-MSCs and mBM-MSCs were cultured in osteogenic induction medium for four cycles, followed by (a) alkaline phosphatase staining (magnification, ×100; scale bar, 100 µm) and (b) RT-qPCR analysis of mRNA levels of Runx2 (***P<0.001). mUC, mouse umbilical cord; mBM, mouse bone marrow; MSCs, mesenchymal stem cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; Runx2, runt-related transcription factor 2.

Figure 4.

TLR3-specific ligand poly(I:C) increased the expression of stemness-related proteins in mUC-MSCs. (A) Reverse transcription-polymerase chain reaction analysis of the expression of TLR3 in mUC-MSCs. (B) Western blot analysis for the expression levels of Sox2, Nanog and Oct4 in mUC-MSCs pre-treated with 0, 10, 25 and 50 µg/ml poly(I:C) for 24 or 48 h. mUC, mouse umbilical cord; mBM, mouse bone marrow; MSCs, mesenchymal stem cells; poly(I:C), polyinosinic:polycytidylic acid; TLR3, Toll-like receptor 3; Sox2, Sex determining region Y-box 2; Oct4, octamer-binding transcription factor 4.

Figure 5.

Poly(I:C) induces cytokine production in mUC-MSCs. A luminex assay of the levels of 10 cytokines in the supernatants from mUC-MSCs pre-treated with poly(I:C) for (A) 24 h or (B) 48 h, including G-CSF, IFN-γ, IL-1β, IL-6, IL-10, IL-15, IL-17, CXCL10, VEGF and TNF-α. (C) Reverse transcription-quantitative polymerase chain reaction analysis of mRNA levels of IL-6, IL-8, CCL5 and CXCL10 in mUC-MSCs treated with poly(I:C) for 24 or 48 h. ***P<0.001 and **P<0.01, vs. control group. mUC-MSCs, mouse umbilical cord mesenchymal stem cells; G-CSF, granulocyte colony stimulating factor; IFN-γ, interferon-γ; IL, interleukin; CXCL10, chemokine (C-X-C motif) ligand 10; VEGF, vascular endothelial growth factor; TNF-α, tumor necrosis factor-α; CCL5, chemokine (C-C motif) ligand 5; poly(I:C), polyinosinic:polycytidylic acid.