Malignant tumor formation after transplantation of short-term cultured bone marrow mesenchymal stem cells in experimental myocardial infarction and diabetic neuropathy

Abstract

Rationale: Bone marrow (BM)-derived mesenchymal stem cells (MSCs) hold great promise for cardiovascular cell therapy owing to their multipotency and culture expandability.

Objective: The aim of the study was to investigate whether MSCs can treat experimental acute myocardial infarction (MI) and diabetic neuropathy.

Methods and results: We isolated mononuclear cells from mouse BM and cultured MSCs in a conventional manner. Flow cytometry analyses of these cultured cells at passage 4 showed expression of typical MSC markers such as CD44 and CD29, but not hematopoietic markers such as c-kit, flk1, and CD34. To determine the therapeutic effects of MSCs, we injected MSCs into the peri-infarct area after ligation of the left anterior descending coronary arteries of mice and, as separate experiments, injected the same batch of MSCs into hindlimb muscles of mice with diabetic neuropathy. During the follow-up at 4 to 8 weeks after cell transplantation, growing tumors were observed in 30% of hearts in the MI model, and in 46% of hindlimbs in the diabetic neuropathy model. Histological examination of the tumors revealed hypercelluarity, pleomorphic nucleoli, cytological atypia and necrosis, and positive staining for α-smooth muscle actin, indicative of malignant sarcoma with myogenic differentiation. Chromosomal analysis of these MSCs showed multiple chromosomal aberrations including fusion, fragmentation, and ring formation.

Conclusions: Genetically unmodified MSCs can undergo chromosomal abnormalities even at early passages and form malignant tumors when transplanted in vivo. These results suggest that careful monitoring of chromosomal status is warranted when in vitro expanded MSCs are used for cell therapy such as for MI.

Figure 1. Transplanted MSCs generated malignant tumors in a mouse model of diabetic neuropathy

A, Representative tumors (arrows) in the hindlimbs following BM-MSC transplantation into hindlimb muscles. B, Serial cross sectional images of the tumors showed multiple sites of necrosis (arrowheads). C–F, H & E staining showed fascicular arrangement of pleomorphic spindle cells and area of geographic necrosis (C, an arrowhead). The tumor cells showed nuclear hyperchromatism, pleomorphism, atypical mitosis (D, double arrows), and increased mitotic figures (E, arrows) suggesting pleomorphic sarcoma. The tumor cells were elongated and had abundant pinkish cytoplasms (F). The nuclei were centrally located and blunt ended. Original magnification ×400. Scale bar, 100 µm. G, Immunohistochemistry with α-smooth muscle actin showed that tumors were focally positive for α-smooth muscle actin, which is compatible with the pleomorphic sarcoma with myogenic differentiation. H, Positive control of α-smooth muscle actin staining (scale bar, 100 µm).

Figure 1. Transplanted MSCs generated malignant tumors in a mouse model of diabetic neuropathy

A, Representative tumors (arrows) in the hindlimbs following BM-MSC transplantation into hindlimb muscles. B, Serial cross sectional images of the tumors showed multiple sites of necrosis (arrowheads). C–F, H & E staining showed fascicular arrangement of pleomorphic spindle cells and area of geographic necrosis (C, an arrowhead). The tumor cells showed nuclear hyperchromatism, pleomorphism, atypical mitosis (D, double arrows), and increased mitotic figures (E, arrows) suggesting pleomorphic sarcoma. The tumor cells were elongated and had abundant pinkish cytoplasms (F). The nuclei were centrally located and blunt ended. Original magnification ×400. Scale bar, 100 µm. G, Immunohistochemistry with α-smooth muscle actin showed that tumors were focally positive for α-smooth muscle actin, which is compatible with the pleomorphic sarcoma with myogenic differentiation. H, Positive control of α-smooth muscle actin staining (scale bar, 100 µm).

Figure 2. Transplanted MSCs generated malignant tumors in a mouse model of acute myocardial infarction

A, Gross necropsy examination after opening the chest wall showed that the tumor mass (arrow) is extended to pericardial sac and invaded into the lung and chest wall. B and C, The explanted tumor showed infiltrative growth into the myocardium, pericardium and chest wall. D, Light microscopic examination after H & E staining revealed infiltrative growth of hypercellular tumor surrounding almost one third of the heart and protruding out of heart. The tumor showed fascicular arrangement of atypical spindle cells having pleomorphic, hyperchromatic nuclei and eosinophilic cytoplasm. Black scale bar 1000 µm. White scale bar 100 µm. E and F, IHC with α-smooth muscle actin showed the tumor was focally positive for α-smooth muscle actin staining. Green fluorescence, α-smooth muscle actin; Blue fluorescence, DAPI for nuclei. G, Positive control of α-smooth muscle actin staining (scale bar 100 µm).

Figure 3. Characterization of tumor-forming MSCs

A, MSCs showed typical spindle-shaped morphology. Scale bar 100 µm. B, FACS analysis showed that 99% of these MSCs expressed CD44 and CD29, but not c-kit, CD31, and CD34. C, Chromosome analyses of normal MSCs showed normal karyotype, 40 XY. D Chromosome analyses of tumor-forming MSCs demonstrated multiple chromosomal abnormalities including fusion, fragmentation, and ring formation.