Additional Supporting Information may be found in the online version of this article.

STEM_764_sm_supplFigure1.TIF434KS1: Schematic illustration of the experimental design and beneficial effects of De-MSCs in HIBD model.
STEM_764_sm_supplFigure2.TIF1703KS2: Neuronal differentiation of MSCs in vitro. Monoclonal MSCs (clone 3) were plated at 10x102/cm2 in 6 well plates and neuronal differentiation was performed as described in Materials and Methods. a: Phase contrast photographs of neuronal differentiation at different time points (24-72 hours). scale bar= 100μm b: RT-PCR analysis for selected markers of lineage commitment at successive time-points of MSC differentiation. c-f: Quantitative analysis of b, data were from three independent experiments. **, p<0.01; ***, p<0.001.
STEM_764_sm_supplFigure3.TIF1683KS3: Different clones of MSCs showed the same enhanced neuronal potential and survival after dedifferentiation process. Monoclonal MSCs (clone 1, 2, 3, 5) were plated at 10x102/cm2 in 6 well plates and neuronal differentiation (24 hours), dedifferentiation (24 hours) and re-differentiation (24 hours) were performed as described in Materials and Methods. a: Flow cytometry analysis of MSCs (clone 1 and clone 2) showing similar stem cell surface markers. b: Phase contrast photographs of neuronal differentiation, dedifferentiation and re-differentiation of clone 1 and clone 2 MSCs □scale bar= 100μm. c: Immunostaining of the general neuronal markers, NF-M during neuronal differentiation, dedifferentiation and re-differentiation process for clone 2 MSCs. (Scale bar=100μm). d: Quantification analysis showing enhanced neuronal differentiation after dedifferentiation process in different clones. *, p<0.05; **, p<0.01. e: MTT analysis showing enhanced cell survival upon different concentration of H2O2 in dedifferentiated clone 2 MSCs, *, p<0.05.
STEM_764_sm_supplFigure4.TIF840KS4: Concomitant upregulation and reversion of multiple neurogenesis marker genes as determined by PCR array focused on neurogenesis and neural stem cells (Qiagen, PARN-404c). The expression levels of multiple neurogenesis genes were markedly increased in differentiated cultures (24 hours) compared with undifferentiated MSCs. In addition, dedifferentiation (24 hours) from the neuronal to the stem cell phenotype was associated with a marked reduction in the expression of neuronal markers. Furthermore, De-MSCs could undergo redifferentiation (24 hours) with full expression of the neuronal markers, indicating an enhanced neuronal potential in De-MSCs.
STEM_764_sm_supplFigure5.TIF665KS5: Molecular characterization of De-MSCs. a: RT-PCR analysis confirmed the expression profiles of some altered genes indicated in the genome microarray. b: Immunofluoresence staining of Tri-Methyl-H3K4 and Tri-Methyl-H3K27 in MSCs, MSC-differentiated neurons and De-MSCs, with corresponding DAPI staining and merged images (scale bar= 30 μm).
STEM_764_sm_supplFigure6.TIF813KS6: Enhanced cell proliferation was not retained in De-MSCs after passaging. a: Representative immunofluorescence staining of PCNA. 1x104 MSCs or De-MSCs at passage 2 (5 days after dedifferentiation) were seeded on individual coverslide in 6 well plate. 24 hours after, the cells were stained with PCNA as described in Materials and Methods, scale bar=100μm. b: Quantitative analysis of a, *, p<0.05. c: Flow cytometric analysis of cell cycle progression in MSCs and De-MSCs showing no significant difference between MSCs and De-MSCs cultured for 48hours.
STEM_764_sm_supplFigure7.TIF1333KS7: De-MSCs with advanced passage remained their advantage in cell survival and neuronal differentiation. a: Flow cytometry analysis of CD markers for De-MSCs at passage 3 (day 10 after dedifferentiation). (a'), CD34; (b'), CD45; (c'): CD29; (d'): CD90. b: MTT assay showing De-MSCs at passage 3 (day 10 after dedifferentiation) retained enhanced cell survival compared to undifferentiated MSCs. *, p<0.05; **, p<0.01. c: FACS analysis of Annexin- V/propidium iodide-stained cells in MSCs and De- MSCs at passage 3 (p3, day 12 after dedifferentiation) challenged with 250μM H2O2 in media containing 0.5% FBS for 24 hours. Primary FACS plots were shown in the upper panel (a': MSCs; b': MSCs with H2O2; c': De-MSCs p3; d': De-MSCs p3 with H2O2) and quantifications from two independent experiments were shown in the lower panel **, p<0.01. d: Immunofluoresence staining of NF-M in MSCs, De-MSCs at passage 3 (day 12 after dedifferentiation), and De-MSCs (p3) induced reneuron with corresponding DAPI staining and merged images (scale bar= 100 μm).
STEM_764_sm_supplFigure8.TIF393KS8: MSCs and De-MSCs (24 hours after dedifferentiation) were serum starved for 24 hours and changes of bcl-2 family proteins were detected by western blot.
STEM_764_sm_supplFigure9.TIF2003KS9: Phase contrast images showing that some MSCs and De-MSCs (24 hours after dedifferentiation) rapidly transformed from fibroblastic to neuronal morphology 24 hours after co-culture with H2O2 treated PHN.
STEM_764_sm_supplFigure10.TIF1530KS10: Immunohistochemical staining of MAP2 on day 3 or day 7 brain sections of HIBD rat implanted with MSCs or De-MSCs (24 hours after dedifferentiation) (scale bar=20μm).
STEM_764_sm_supplFigure11.TIF397KS11: Schematic illustration of the shuttle box test.
STEM_764_sm_supplFigure12.TIF277KS12: Gene ontology analysis showing significantly different biological processes between De-MSCs (24 hours after dedifferentiation) and MSCs treated HIBD brains. One week after transplantation, the HIBD brains were collected and RNA was extracted and hybridized on Agilent whole rat genome oligo microarray as described in Materials and Methods.
STEM_764_sm_suppldata.pdf102KSupplementary Data
STEM_764_sm_supplTable1.pdf58KSupplementary Table 1

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