GFP expression in PCs and putative interneurons after grafting hfMSCs carrying the inverted GFP gene to the SCA1 mouse cerebellum.

(a) Schema depicting the FLEx-Tet system, which permitted us to explore the fusion of hfMSCs with cerebellar neurons. An AAV9-SynImCMV-HA-mtTA-P2A-Cre vector expressing HA-tagged mtTA and Cre through a neuron-specific SynImCMV promoter was injected into the cerebella of 6–8-month-old SCA1 mice (1). A sequence with TRE and inverted GFP genes flanked by loxP and lox2272 was inserted into the genome of hfMSCs by using Lenti-TRE-FLEx-GFP vectors (2). Thus, GFP protein was never produced without Cre recombinase, and the expression increased drastically after co-provision with mtTA. The TRE-FLEx-GFP-hfMSCs (50,000 cells) were then injected into the SCA1 mice 2 weeks after injection of AAV9-SynImCMV-HA-mtTA-P2A-Cre vectors (3). (b-g) Emergence of GFP-expressing neurons in the cerebella of mice that were sacrificed 2 weeks after the TRE-FLEx-GFP-hfMSC injection. The cerebellar slices were immunolabeled for GFP and calbindin, a PC marker. GFP expression was detected in PCs (b, e-g) and cells in the molecular layer (c, d). Arrows in (e-g) indicate a GFP-expressing PCs co-immunostained for calbindin. Because we used a neuron-specific synapsin I promoter with a minimal CMV sequence, Cre and mtTA should have been expressed specifically in neurons, and thus the GFP-expressing cells in the molecular layer (c, d) were presumed to be basket cells (c) and stellate cells (d). Scale bar, 50 μm.