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Mash1 and Neurogenin 2 Enhance Survival and Differentiation of Neural Precursor Cells After Transplantation to Rat Brains via Distinct Modes of Action
Sang-Hoon Yi, A-Young Jo, Chang-Hwan Park, Hyun-Chul Koh, Rae-Hee Park, Haeyoung Suh-Kim, Incheol Shin, Yong-Sung Lee, Jaesang Kim and Sang-Hun Lee
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Morphologic maturation of the neurons differentiated from NPCs expressing the proneural bHLH transcription factors Ngn2 and Mash1. NPCs from E14 rat cortices were plated at 2000 cells/6 cm-dish, transduced as indicated, and subjected to TuJ1-immunostaining 3 days after differentiation. Shown in a-c are representative images of TuJ1-positive neurons in the cultures transduced with LacZ- (a), Ngn2- (b), and Mash1 (c). Scale bar, 20 
m. Graph d depicts the total length of TuJ1+ fibers per cell.
Significantly different from the LacZ-control(*) or Mash1-transduced cells(#) at p<0.001.
Figure S2. (jpeg 79K)
Ngn2 and Mash1 effects on neuronal yield and cell growth of NPCs isolated from various regions and days of rat embryonic brains. NPCs were isolated and cultured from different embryonic ages (E13-E15) and regions (cortex, ventral parts of the midbrain, hindbrain, and spinal cord), and transduced with retroviruses expressing Ngn2, Mash1, or LacZ (control) as described in Materials and Method. TuJ1+ (a), MAP2+ (b) neuron yields and viable cell numbers (c) were counted 3 days after differentiation in vitro. The graphs represent % immunoreactive cells out of total cells (a-b) and % values of viable cell counts relative to the respective LacZ-controls (c). Significantly, different from the LacZ-control (*) at p<0.001.
Figure S3. (jpeg 92K)
Semi-quantitative RT-PCR analyses for the ligands and receptors involved with signaling pathways specific to NPC proliferation including FGF-, EGF-, LIF-, Wnt-, Notch-, and SHH-signaling pathways. The expression analyses were carried out using the cultures 2 days after Ngn2-, Mash1-, and LacZ-transductions.
Figure S4. (jpeg 97K)
In vivo survival and neuronal differentiation of Ngn2 or Mash1-transduced NPCs grafted in adult rat hippocampus. To label donor cells with GFP, NPCs derived from E14 rat cortices were transduced with the viruses carrying the bicistronic vectors pLacZ-IRES-GFP, pMash1-IRES-GFP, or pNgn2-IRES-GFP. The transduced NPCs were transplanted into the rat hippocampus as indicated. (a-c) Representative confocal image stacks (35 m in the z direction) of the NeuN+/GFP+ striatal grafts at 2 weeks after transplantation of NPCs transduced with LacZ (a), Ngn2 (b), and Mash1 (c). Scale bar, 80 m. (d-e) Quantification of graft volumes (d), NeuN+ neurons in graft (e) at 2 weeks post-transplantation.
Figure S5. (jpeg 91K)
Absence of prolonged cell proliferation in Mash1-transduced precursors. (a) In vitro cell growth pattern of Mash1-transduced NPCs after 9 days of differentiation. (b-e) Representative images of the cells positive for the M-phase marker pHH3 in the striatal grafts generated by LacZ (b)-, Mash1 (c)-, and Ngn2 (d)-transduced NPCs at 2 weeks after transplantation. Shown in e are pHH3+ cells in the striatum of the rat brain grafted with NPCs derived from human embryonic stem cells (hES-NPCs) 8 weeks after transplantation. hES-NPCs were generated and transplanted as described by Ko et al., 2007. Note that none or very few cells were positive for pHH3 in b-d, whereas pHH3+ cells were relatively abundant in the hES-NPC-derived graft (e). The borders of the grafts are indicated by dashed lines. Insets are high magnification images of the respective boxed areas. Scale bar, 80 m.
