Isolation and characterization of a novel strain of mesenchymal stem cells from mouse umbilical cord: potential application in cell-based therapy

Abstract

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have recently been recognized as a potential source for cell-based therapy in various preclinical animal models, such as Parkinson's disease, cerebral ischemia, spinal cord injury, and liver failure; however, the precise cellular and molecular mechanisms underlying the beneficial outcomes remain under investigation. There is a growing concern regarding rejection and alteration of genetic code using this xenotransplantation approach. In this study, a novel strain of murine MSCs derived from the umbilical cord of wild-type and green fluorescent protein (GFP) transgenic mice have been successfully isolated, expanded, and characterized. After 10 passages, the mUC-MSCs developed a rather homogeneous, triangular, spindle-shaped morphology, and were sub-cultured up to 7 months (over 50 passages) without overt changes in morphology and doubling time. Cell surface markers are quite similar to MSCs isolated from other tissue origins as well as hUC-MSCs. These mUC-MSCs can differentiate into osteoblasts, adipocytes, neurons, and astrocytes in vitro, as well as hematopoietic lineage cells in vivo. mUC-MSCs also possess therapeutic potential against two disease models, focal ischemic stroke induced by middle cerebral artery occlusion (MCAo) and acute hepatic failure. Subtle differences in the expression of cytokine-related genes exist between mUC-MSCs and hUC-MSCs, which may retard and jeopardize the advance of cell therapy. Allografts of these newly established mUC-MSCs into various mouse disease models may deepen our insights into the development of more effective cell therapy regimens.

Figure 1. Isolation of novel MSCs from mouse umbilical cord.

(A) Mouse UC-MSCs isolated from GFP transgenic mice at passage numbers 0 (Div 4) and 10 (P10). mUC-MSCs displayed the triangular, spindle-shaped, and fibroblast-like morphology at P10. (B) Growth Kinetics of mUC-MSCs. Cumulative cell number was counted at each passage. (C) The population doubling time pattern of mUC-MSCs from passage 10 to passage 50. (D) RT-PCR analysis of the pluripotency-associated genes in mUC-MSCs. Lane 1, mES cells; lane 2, germ cells from testis; lane 3, mUC-MSCs from heterogeneous populations (Mix); land 4-5, mUC-MSCs derived from different single colony. Scale bar = 50 µm.

Figure 2. Immunophenotype antigen profile of mUC-MSCs.

Cells of passage number 12–20 were labeled with PE- or APC-conjugated antibodies against the indicated antigens, and analyzed by flow cytometry. Hematopoietic cells markers, including CD2, CD3, CD5, CD11b, CD19, CD45, CD117, Gr-1, and TER-119; mesenchymal stem cells markers, including CD13, CD29, CD44, CD49e, and Sca-1; and mES cells marker, SSEA-1, were used. The respective isotype control was displayed as an open histogram (dark gray line), and specific antibody was displayed as a filled histogram (green).

Figure 3. Mesodermal differentiation potential of mUC-MSCs.

Osteogenic differentiation: Cells of passage number 12–20 were induced to form osteoblast by culturing in osteogenic induction medium for 28 days, followed by staining with Alizarin Red-S (A), von Kossa (B), and evaluated by alkaline phosphatase activity (C) for osteoblasts. Adipogenicdifferentiation : cells were induced to form adipocytes by culturing in induction medium for 14 days. Adipocytic differentiation is evidenced by the formation of oil droplets stained with Oil-red O. Panel D (right) displayed a higher-magnification image of cells. (E) Expression of adipocytic phenotypic markers PPAR-γ1 and PPAR-γ2 was assayed by RT-PCR. Scale bar = 50 µm.

Figure 4. Neuroectodermal differentiation potential of mUC-MSCs.

Cells of passage number 12–20 were induced to form neural cells by culturing in induction medium for 7 days. Cells were co-stained with DAPI and neural progenitor marker, nestin, at 6 hr (A); or astroglial marker, GFAP (B); and neuronal markers, including MAP 2 (C) and βⅢ-tubulin (D), at 5 days post-induction. Scale bar = 50 µm.

Figure 5. mUC-MSCs transplantation improves mice survival after hematopoietic reconstitution.

(A) The C57BL/6JNarl mice were subjected to a lethal dose of 10 Gy γirradiation, and administered 5 × 10⁶ cells of GFP-mUC-MSCs (n = 6) or PBS control (n = 7) via tail vein injection 24 hr later. The survive rate of the recipient mice was analyzed. (B) The expression of GFP in liver, spleen, bone marrow (BM), and peripheral blood (PB), was analyzed 6 months post-transplantation by genomic DNA PCR. The expression of IL-2 serves as internal control. (C) The C57BL/6JNarl mice were subjected to a sub-lethal dose of 8 Gy γ-irradiation and administered with GFP-mUC-MSCs (n = 18) or PBS control (n = 18) via tail vein injection 24 hr later. (D) The hematopoietic lineage cells (Lin⁺ cells) of peripheral blood from mice (n = 6, each group) in (C) were analyzed 6 weeks after sub-lethal irradiation. The isolated Lin⁺ cells were subjected to further extraction of genomic DNA and GFP expression was analyzed by PCR. PC = positive control, the tissues or Lin⁺ cells from GFP-transgenic mice; NC = negative control, the tissues or Lin⁺ cells from wild-type C57BL/6JNarl mice; MSC = GFP-mUC-MSCs.

Figure 6. mUC-MSCs transplantation attenuates ischemic brain injury.

(A) The C57BL/6JNarl mice were administrated 4 × 10⁵ cells of mUC-MSCs, hUC-MSCs, or PBS control at 1 day after a 120-min MCAo. Infarct volume was evaluated 14 days later. (B) Quantification of infarct volume in mice with mUC-MSCs (n = 7), hUC-MSCs (n = 7), or PBS control (n = 7) treatment. For assessment of differentiation potential of mUC-MSCs in vivo, mUC-MSCs were pre-labeled with Cell Tracker CM-DiI fluorescent dye and co-stained with specific cellular markers, including endothelial cell marker, CD31, at day 3 (C); or neural progenitor marker, nestin; neuron marker, NeuN; astroglial marker, GFAP; and macrophage/microglia marker, CD11b at day 7 (D–G) after MCAo. Data shown here are displayed as the mean ± SEM. **P < 0.01, ***P < 0.001 versus PBS control.

Figure 7. mUC-MSCs transplantation improves mice survival after TAA-induced acute hepatic failure.

Male C57BL/6JNarl mice were administrated TAA (1000 mg/kg intraperitoneal injection) 6 hr prior to receiving 2 × 10⁶ cells of mUC-MSCs (n = 17) or PBS control (n = 15) via tail vein injection, and the survival rate was analyzed.