Fusion between human mesenchymal stem cells and rodent cerebellar Purkinje cells

Abstract

Aims: we explored whether cellular fusion and heterokaryon formation between human and rodent cells in the cerebellum of mice occurs after intravenous injection of human bone marrow-derived mesenchymal stem cells (MSCs). The influence of central nervous system inflammation on this process was also assessed. In addition, we examined whether tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma, factors associated with inflammation, increase cellular fusion between human MSCs and rodent cerebellar neurons in vitro.

Methods and results: human MSCs were intravenously injected into mice with experimental autoimmune encephalomyelitis (EAE) and control mice. After 22 days, mouse Purkinje cells expressing human Golgi Zone were found within the Purkinje cell layer of the cerebellum, indicating that fusion and heterokaryon formation had occurred. The numbers of heterokaryons in the cerebellum were markedly increased in mice with EAE compared with control mice. Rodent cerebellar neuronal cells labelled with enhanced green fluorescent proteinin vitro were co-cultured with human bone marrow-derived MSCs in the presence of TNF-alpha and/or IFN-gamma to determine their influence on fusion events. We found that fusion between MSCs and cerebellar neurons did occur in vitro and that the frequency of cellular fusion increased in the presence of TNF-alpha and/or IFN-gamma.

Conclusions: we believe that this is the first paper to define fusion and heterokaryon formation between human MSCs and rodent cerebellar neurons in vivo. We have also demonstrated that fusion between these cell populations occurs in vitro. These findings indicate that MSCs may be potential therapeutic agents for cerebellar diseases, and other neuroinflammatory and neurodegenerative disorders.

Figure 1

Mesenchymal stem cell cultures display a typical MSC phenotype and can be induced to differentiate towards osteogenic, adipogenic and chondrogenic lineages. (A) Flow cytometric analysis of CD29, CD105, CD166, CD44 and CD45 expression by mesenchymal stem cell (MSC) cultures at third passage (n = 4). Images depicting MSC cultures, at third passage, differentiated down osteogenic, adipogenic and chondrogenic lineages. Osteogenic differentiation was visualized by the presence of high levels of alkaline phosphatase (B) and using immunofluorescent detection of Alkaline phosphatase (red)/nuclei (blue) (C). Chondrogenic differentiation was characterized by Alcian blue staining (D) and the immunofluorescent detection of aggrecan (red)/nuclei (blue) (E). Adipogenic differentiation was visualized by the accumulation of lipid-containing vacuoles which stain red with oil red O (F) and using immunofluorescent detection of lipoprotein lipase (red)/nuclei (blue) (G).

Figure 2

Anti-human Golgi Zone binds specifically to human mesenchymal stem cell (MSC) and not rodent cells. Images are of rat E18 cerebellar neuronal (A/B) and human MSC (C/D) cultures co-labelled with human Golgi Zone (red), βIII tubulin (green) and Hoescht nuclear stain (blue) (A&C are images of respective negative controls using no primary antibodies).

Figure 3

Mouse Purkinje cells stain for human Golgi Zone within the cerebellum of Naïve/experimental autoimmune encephalomyelitis (EAE)-mice treated with intravenous human mesenchymal stem cells (MSCs). Immunofluorescence photographs (a), (b) and (c) refer to dashed areas a, b, c in image (C). A cerebellum section from an EAE-mouse treated with intra-venous human MSCs immunologically labelled with human Golgi Zone (A) (red) and Calbindin-D28K (B) (green) and, merged images (C). (D) A diagram of a typical and complete area of a sagittally sectioned mouse cerebellum in which the presence of human MSCs was investigated and numerated.

Figure 4

An increase in mouse Purkinje cells expressing human Golgi Zone in experimental autoimmune encephalomyelitis (EAE) vs. naïve mice treated with intra-venous human MSCs. (A) Quantification of Purkinje cells co-expressing human Golgi Zone in naïve mice (n = 5) and EAE mice (n = 5) 22 days post MSC infusion (**P < 0.01). (B) Quantification of the mean number of Purkinje cells per random field within the cerebellum of EAE and naïve mice (n = 5) 22 days post MSC infusion.

Figure 5

Human mesenchymal stem cells (MSCs) fuse with rat cerebellar enhanced green fluorescent protein (EGFP)-transduced neurons in vitro. Confocal images are of rat EGFP-transduced E18 cerebellar neuronal/human MSC co-cultures immunologically labelled with Hoescht nuclear stain (blue; a), EGFP (green; b), human Golgi Zone (red; c) and merged image (d). (A) Low power figure; arrows indicate dual stained EGFP/human Golgi Zone (red/green) cells; (B) high power figure; arrow head (human Golgi Zone positive/EGFP negative MSC), arrow head and star (human Golgi Zone negative/EGFP positive neuron), arrow (human Golgi Zone positive/EGFP positive fused MSC/neuronal cell). (C) An enlarged area of the dashed box in (B) showing a bi-nucleated human Golgi Zone positive/EGFP positive fused MSC/neuronal cell.

Figure 6

The effect of imflammatory cytokines tumour necrosis factor (TNF)-alpha/interferon (IFN)-gamma on human mesenchymal stem cell (MSC)/rat cerebellar neuronal fusion in vitro (*P < 0.05, **P < 0.01, n = 4). Co-cultured in base medium (Base); TNF (TNF-alpha 5 ng/ml), IFN (IFN-gamma 5 ng/ml) or TNF-alpha and IFN-gamma (5 ng/ml). (A) The number of enhanced green fluorescent protein (EGFP) positive neurons per field, (B) the number of Golgi Zone (GZ) positve MSCs per field, (C) the number of EGFP/GZ positive fused cells per field and (D) the percentage of EGFP positive neurons co-expressing human GZ for each experimental condition.