1. Volume 4, Issue 2, Pages 271-286 (July 2013)
Wnt/β-Catenin Signaling Triggers Neuron Reprogramming and Regeneration in the Mouse Retina 
Daniela Sanges, Neus Romo, Giacoma Simonte, Umberto Di Vicino, Ariadna Diaz Tahoces, Eduardo Fernández, Maria Pia Cosma 
Cell Reports 
Volume 4, Issue 2, Pages (July 2013)
DOI: /j.celrep
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2. Cell Reports 2013 4, 271-286DOI: (10.1016/j.celrep.2013.06.015)
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3. Figure 1 NMDA Retinal Damage Induces Cell Fusion In Vivo
(A) Schematic representation of the cell fusion experimental plan. In vivo cell fusion between HSPCsRFP/Cre and retinal neurons of LoxP-STOP-LoxP-YFP mice (R26Y) leads to excision of a floxed stop codon in the retinal neurons and, in turn, to expression of YFP. The resulting hybrids express both YFP and RFP.
(B) Confocal photomicrographs of flat-mounted retinas focused on the gcl of R26Y NMDA-damaged (NMDA) or healthy (control) eyes of mice transplanted with HSPCsRFP/Cre harvested 24 hr after transplantation. Double-positive RFP (red) and YFP (green) hybrids derived from cell fusion are detected in the presence of NMDA damage (NMDA) but not in the undamaged eye (control). Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.
(C–E) Immunohistochemical analysis of the retinal fusion cell partners. YFP+ hybrids positive for Müller glia (C, GS, red), amacrine (D, Chat, red) or ganglion (E, SMI-32, red) cell markers are detected 24 hr after transplantation of HSPCsCre in NMDA-damaged eyes. Yellow arrows indicate cells positive to both YFP and marker staining. Scale bar: 50 μm.
See also Figures S1, S2, and S3.
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4. Figure 2
Activation of the Wnt Signaling Pathway Enhances Neuron Reprogramming after HSPC Fusion In Vivo
(A) Gene expression profile of fluorescence-activated cell sorted RFP+/YFP+ hybrids after transplantation of untreated (No BIO, white bars) and BIO-treated (black bars) HSPCsCre/RFP in R26Y NMDA-damaged eyes. Data are real-time PCR quantifications plotted as the fold change with respect to controls from complementary DNAs of NMDA-damaged retinas mixed with either untreated HSPCs or BIO-treated HSPCs. Data are mean ±SEM (n = 6).
(B and E) Schemes of cell-fusion-mediated reprogramming to identify Nestin (B) or Nanog (E) promoter activation in transgenic mice.
(C and F) Double-red+/green+-reprogrammed hybrids were detected only in the presence of NMDA damage in flat-mounted retinas after transplantation of HSPCsR26Y/RFP in Nestin-Cre (C) and HSPCsRFP in Nanog-GFP (F) eyes. Scale bars: 50 μm.
(D and G) Quantification of double-red+/green+ hybrids activating Nestin (D) and Nanog (G) promoters evaluated according to Wnt signaling modulation. Undamaged or NMDA-damaged retinas treated or not with DKK1 were transplanted after 24 hr with either untreated, Wnt3a-treated (Wnt3a), or BIO-treated (BIO) HSPCs. Data are mean ±SEM (n = 90) counted in vertical sections (as for Figure S2B). ∗∗∗p <
(H and I) Mouse (H) and human (I) gene expression profiles of fluorescence-activated cell sorted DiD+/GFP+ hybrids after transplantation of DiD-labeled human CD34+ HSPCs in NMDA-damaged eyes of Nanog-GFP mice. Data are mean ±SEM (n = 9).
See also Figures S4, S5, S6, S7, and S8.
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5. Figure 3 Reprogrammed Hybrids Can Proliferate and Differentiate
(A) Quantification of mitotic (Ki-67+) and apoptotic (Annexin V+) YFP+ hybrids after transplantation of untreated (No BIO) or BIO-treated (BIO) HSPCsCre in NMDA-damaged R26Y eyes. Data are mean ±SEM; n = 30. ∗∗∗p <
(B–E) Ki67 (B and D, red) and Annexin V (C and E, red) staining for YFP+ (green)-reprogrammed hybrids obtained 24 hr after injection of BIO-treated (BIO-HSPCs) or untreated-HSPCsCre in NMDA-damaged R26Y eyes. Nuclei were counterstained with DAPI (blue).
(F and G) Quantification of YFP+ hybrids positive to different markers, as counted after immunostaining of sections (as for Figure S2B) at different times after transplantation of BIO-treated (F) and untreated (G) HSPCsCre into NMDA-damaged eyes of R26Y mice. Data are mean ±SEM (n = 30).
(H) YFP+ hybrids (green) obtained 24 hr after transplantation of BIO-treated HSPCsCre in NMDA-damaged R26Y eyes stained with Oct4, Nanog, and Nestin antibodies (red). Nuclei were counterstained with DAPI.
Scale bars: 50 μm (B, C, D, E, and H).
See also Figure S9.
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6. Figure 4
Long-Term Differentiation Potential of Hybrids Obtained after Cell-Fusion-Mediated Reprogramming
(A) Experimental strategy used to identify YFP+ hybrids 1 month after BIO-treated or untreated HSPCsCre in NMDA-damaged R26Y retinas.
(B) YFP+ neurons were detected in NMDA-injured R26Y retinal flat mounts 1 month after BIO-HSPCsCre transplantation. Nuclei were counterstained with DAPI (blue). Right: higher magnification of the YFP+ neurons.
(C) YFP+ differentiated hybrids (green) expressing either the ganglion cell marker SMI-32 (left, red) or the amacrine cell marker Chat (right, red).
(D) YFP+ axons (green) detected in optic nerves from eyes transplanted with BIO-HSPCsCre but not with untreated HSPCsCre. Right: higher magnification of the YFP+ axons (green) in the optic nerve.
Scale bars: 50 μm (B, C, and D) or 200 μm (D).
See also Figure S10.
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7. Figure 5
NMDA-Damaged Retinas Can Be Regenerated after Fusion of Transplanted BIO-Treated HSPCs
(A and B) Evaluation of the percentage of YFP+ (green) hybrids also positive to SMI-32 staining (red) in flat-mounted retinas harvested 24 hr or 1 month after transplantation of BIO-treated (BIO, black bars) or untreated (No BIO, gray bars) HSPCsCre in NMDA-damaged R26Y eyes (n = 3). Twenty different random fields were analyzed for each retina. A representative image of regenerated ganglion cells (yellow arrows) double positive for YFP (green) and SMI-32 (red) is shown in (B). Scale bar: 50 μm.
(C) Hematoxylin and eosin staining showing increases in thickness of the inner nuclear layer (inl, square brackets) and regeneration of the ganglion cell layer (gcl, arrowheads) in NMDA-damaged retina 1 month after BIO-HSPCsCre transplantation. Transplantation of untreated HSPCsCre or BIO alone does not induce regeneration. Arrows indicate ganglion cell loss in the NMDA-damaged retinas. Arrowheads indicate ganglion cell nuclei. Scale bars: 50 μm.
(D and E) Quantification of ganglion nuclei in the ganglion cell layer (D, gcl) and nuclear rows in the inner nuclear layer (E, inl) as counted in vertical retinal sections of undamaged or NMDA-damaged retinas treated as in (C). Data are mean ±SEM (n = 30). ∗∗∗p <
(F) All the nuclei excluding those of endothelial cells were counted in the ganglion cell layer along the nasotemporal (left) and dorsoventral (right) axes and plotted as cells per mm2. A total of 80 different images composing the whole retina were counted for each sample. Data are mean ±SEM (n = 3). ∗p < 0.01 between damaged retinas transplanted with BIO-HSPCs and untransplanted damaged retinas. ON, optic nerve.
(G) Total cells in the ganglion cell layer, excluding endothelial cells, counted as in (F), were plotted as density maps, as indicated in the color key.
See also Figure S10.
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8. Figure 6 Multielectrode Recordings from Flat-Mounted Mouse Retinas
(A) Percentage of neurons responding or not responding to light in control retinas, NMDA-damaged retinas, and NMDA-damaged retinas transplanted with BIO-HSPCs. Data are mean ±SEM (n = 5 for each group). ∗∗∗p <
(B) Representative examples of sorted spikes waveforms (top graphs; each color represents a single sorted unit), raster plots of spiking responses, and peristimulus time histograms (PSTHs, bin = 10 ms) (bottom graphs) from typical retinal ganglion cells of control, NMDA-damaged, and NMDA + BIO-HSPC retinas. The light ON/dark bar below the histograms represents the light stimulus that was generated from a computer monitor and was projected onto the retinal surface. ON, ON-OFF, and OFF responses are all recorded by the array. Frequency is in impulses (spikes) per second.
(C) Boxplot latencies to ON peak including (from bottom to top) the smallest observation (sample minimum), lower quartile (25th percentile), median, upper quartile (75th percentile), and largest observation (sample maximum) of each sampled group.
(D) Normalized responses of ON ganglion cells for control, NMDA-damaged, and NMDA + BIO HSPC retinas as a function of stimulus intensity. Each data point shows the mean ±SEM of the percentage of cells responding to each light intensity ranging from black to white (boxes).
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9. Figure 7
Endogenous BM-Derived Cells Recruited in Damaged Eyes Can Fuse with Retinal Neurons
(A) Experimental scheme of endogenous BMC-fusion detection. R26Y mice received BMRFP/Cre transplantation. After BM reconstitution (1 month), the right eyes received an intravitreal injection of NMDA and the left eyes were not injected. The mice were analyzed 24 hr later. Only in the case of cell fusion of recruited BM cells (red) and neurons were YFP/RFP double-positive hybrids detected.
(B) Double-positive YFP/RFP hybrids detected in NMDA-damaged (NMDA) but not in healthy (control) eyes. Scale bar: 50 μm.
(C) Quantification of percentage of YFP+ hybrids with respect to the total number of detected RFP cells (calculated as for Figure S2B). Data are mean ±SEM. ∗∗∗p < 0.001.
(D) Experimental scheme of endogenous cell-fusion-mediated reprogramming. Nestin-Cre mice received BMR26Y transplantation. After BM reconstitution (1 month), the right eyes received an intravitreal injection of BIO + NMDA, while the contralateral eyes were injected with NMDA alone. Nestin-mediated Cre expression leads to expression of the YFP only in the case of cell-fusion-mediated reprogramming of hybrids between recruited BMCsR26Y and neurons.
(E) YFP+ reprogrammed hybrids (green) after fusion of recruited BMCs and damaged neurons were detected only after BIO injection (NMDA + BIO). In contrast, no YFP+ hybrids were seen in NMDA-damaged eyes without BIO (NMDA). Scale bar: 50 μm.
(F and G) Quantification of percentages of mitotic (Ki67+) or apoptotic (Annexin V+) hybrids (F) and Oct4-positive and Nanog-positive hybrids (G), evaluated with the respect to the total number of YFP+ cells. Data are mean ±SEM (n = 30).
See also Figure S11.
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10. Figure S1
Retinal Structure and Analysis of Apoptosis in Retinal Sections, Related to Figure 1
(A) H&E staining (left) of a vertical wild-type retinal section and schematic representation of retinal tissue (right).
(B) TUNEL staining (green) on vertical sections of eyes from R26Y mice sacrificed 24 hr after NMDA injection. Green arrows, apoptotic cells.
(C) NMDA treatment in R26Y eyes does not activate YFP expression (green) in retinal cells. Nuclei were counterstained with DAPI (blue). onl: outer nuclear layer; inl: inner nuclear layer; gcl: ganglion cell layer. Scale bar: 20 μm.
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11. Figure S2 Cell-Cell Fusion Analysis, Related to Figure 1
(A–C) Analysis of fusion efficiency 24 hr after transplantation of HSPCsCre/RFP in NMDA-damaged R26Y eyes. (A) Quantification of RFP+/YFP+ hybrids in vertical sections counted as in described (B). (B) Cell transplantation was performed with at least 3 different eyes for each experiment, and a total of 10 serial sections from each of the eyes were examined in three different regions for each section. The number of YFP+ or GFP+ cells were counted in individual sections within three areas of the retina (40 × optical fields, red rectangles) chosen to include the ganglion cell layer and the inner nuclear layer of the retinal tissue. The ratio between these cells numbers and the total number of red-labeled (DiD) cells or RFP+ cells in the same fields provided the percentages of positive cells (hybrids). (C) FACS analysis of double positive RFP+/YFP+ hybrids detected in NMDA-damaged R26Y retinas transplanted with HSPCsCre/RFP. Non-damaged retinas transplanted with HSPCsCre/RFP were used as control (No NMDA + HSPCsRFP). The percentages of double RFP+/YFP+ cells were also evaluated on the total amount of RFP+ transplanted HSPCs (n = 3). The graph indicates the mean ± s.e.m of three different FACS experiments. Data are means ± s.e.m. (n = 90). ∗∗∗p <
(D) Flow cytometry analysis of tetraploid cells with a 4C content of DNA was performed on gated YFP+ hybrids from NMDA-damaged R26Y retinas transplanted with HSPCCre. Non-transplanted NMDA-damaged retinas were used as control.
(E) YFP+ heterokaryons (arrows) in NMDA-damaged R26Y flat-mounted retinas 24 hr after HSPCsCre transplantation. Nuclei were counterstained with DAPI (blue).
(F and G) FACS analysis of HSPCs fusion with Müller glia and ganglion/ amacrine neurons as percentages of YFP+/DiD+ double-positive hybrids obtained 24 hr after transplantation of DiD-labeled HSPCsR26Y in NMDA-damaged eyes of GFAP-Cre (F, for Müller glia) and CALR-Cre (G, for ganglion/ amacrine neurons) lineage tracing mice.
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12. Figure S3
Cell-Cell Fusion Analysis after Transplantation of ESCs or RSCs, Related to Figure 1
(A) Schematic representation of cell-fusion experimental plan. In-vivo cell fusion between red-labeled (DiD) ESCsCre or RSPCsCre with retinal neurons of R26Y mice leads to the excision of a floxed stop codon in the retinal neurons, and in turn, to the expression of YFP. The resulting hybrids have red membranes and express YFP.
(B) Representative samples of ESCsCre and RSPCsCre injected either into mice eyes pre-treated for 24 hr with NMDA to induce cell damage, or in healthy eyes (control). In R26Y eyes 24 hr after cells injection (DiD cells, red), YFP expression (YFP, green) is detected in the NMDA-damaged eyes (NMDA), but not in the non-damaged eye (control). Nuclei were counterstained with DAPI (blue). onl: outer nuclear layer; inl: inner nuclear layer; gcl: ganglion cell layer. Scale bar: 20 μm.
(C) Quantification of YFP+ cells as percentages relative to the total number of transplanted DiD-positive ESCsCre and RSPCsCre+ in the optical field counted (as for Figure S2B). Sections of NMDA-damaged and non-damaged (control) eyes were analyzed in mice sacrificed 24 hr after transplantation. Data are means ± s.e.m.; n = 30. ∗∗∗p <
(D and E) Analysis of the cell-fusion partners on flat-mounted NMDA-damaged R26Y retinas transplanted with ESCsCre. (D) YFP+ hybrids were also positive either for Müller glia (GS, red), amacrine (Chat, red) or ganglion (SMI-32, red) cell markers. Yellow arrows indicate cells positive to both YFP and marker staining. Scale bar: 10 μm. (E) Quantification of percentages of double marker/YFP positive hybrids evaluated with respect to the total amount of YFP+ hybrids detected as in Figure S2B. Data are means ± s.e.m.
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13. Figure S4
Wnt Pathway Induces In Vivo Cell Fusion-Mediated Reprogramming after HSPC Transplantation, Related to Figure 2
(A) BIO treatment of HSPCs activates Wnt/β-catenin signaling. Real-time PCR quantification of expression of Wnt/β-catenin target gene Axin2 and nuclear translocation of β-catenin.
(B and C) Real-time PCR quantification of different genes in NMDA-damaged R26Y retinas (B) and in BIO-treated (BIO) or untreated (No BIO) HSPCs (C).
(D) NMDA treatment alone does not induce the expression of the Nanog-GFP-puro transgene in treated retinas. Nuclei were counterstained with DAPI (blue).
(E and F) Confocal photomicrographs of vertical sections 24 hr after transplantation of HSPCsRFP or HSPCsR26Y/RFP in undamaged (No NMDA: control) or NMDA-damaged Nanog-GFP (E) and Nestin-Cre (F) eyes. Wnt-signaling after retinal damage was blocked with DKK1 injection (NMDA + DKK1), or activated with HSPCs treatment with Wnt3a for 24 hr in culture before transplantation. Green arrows, GFP+ or YFP+ cells; yellow arrows, double-positive (red+/green+) cells. Nuclei were counterstained with DAPI (blue).
(G) Analysis of double positive RFP+/GFP+ hybrids detected by FACS in NMDA-damaged Nanog-GFP retinas 24h after BIO-treated or untreated HSPCsRFP (No BIO) transplantation. Non-damaged retinas transplanted with HSPCsRFP were used as control (No NMDA: control). The percentages of double RFP+/GFP+ cells were evaluated on the total amount of RFP+ transplanted HSPCs. Data are means ± s.e.m.; n = 3. ∗∗∗p < (H) GFP expression (green) was not detected in retinal sections from healthy Nanog-GFP (no NMDA: control) eyes transplanted with BIO-treated HSPCsRFP. Nuclei were counterstained with DAPI (blue).
Scale bars: 10 μm (A); 50 μm (D, E, F, H). onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer.
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14. Figure S5
Active β-Catenin in Transplanted HSPCs Is Essential to Induce Cell Fusion-Mediated Reprogramming In Vivo, Related to Figure 2
(A and B) Schematic representation of the experimental plan. HSPCs were isolated from double transgenic mice homozygous for a β−catenin allele flanked by flox sites also carrying a Tamoxifen-inducible Cre expression cassette. HSPCs were pre-treated with tamoxifen (TMX) and BIO treated (BIO βcatflox/flox) or not (βcatflox/flox); they were labeled with DiD and transplanted in NMDA-damaged Nanog-GFP eyes. Transplantation of HSPCs that were not treated with tamoxifen (BIO βcat+/+ and βcat+/+) was carried out as control. Formation of double DiD+ (red) and GFP+ (green) reprogrammed hybrids (yellow arrows) was evaluated by confocal photomicrographs on retinal flat mount 24h after transplantation. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.
(C) Real-time PCR quantification of β-catenin expression in HSPCs βcat+/+ and in untreated or BIO- treated HSPCs βcatflox/flox. Data are means ± s.e.m.; n = 3.
(D) BIO-treated or untreated HSPCs βcat+/+ and BIO-treated or untreated HSPCs βcatflox/flox were transplanted in NMDA-damaged Nanog-GFP eyes. The percentage of double DiD+/GFP+ hybrids on the total DiD+ cells detected in the optical fields was counted in vertical sections as for Figure S2B. Data are means ± s.e.m.; n = 3.
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15. Figure S6
Cell Fusion-Mediated Reprogramming after Transplantation of Human CD34+ HSPCs, Related to Figure 2
(A) Confocal photomicrographs of NMDA-damaged Nanog-GFP flat mount retina transplanted with DiD-labeled and BIO-treated human CD34+ HSPCs (red). GFP+/DiD+ hybrids (yellow arrow, green+/red+ cells) were detected. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm.
(B) FACS analysis of double DiD+/GFP+ hybrids obtained 24 hr after transplantation of BIO-treated DiD-labeled hHSPCs in NMDA-damaged Nanog-GFP retinas. Non-damaged transplanted retinas were analyzed as controls.
(C and D) DiD-labeled BIO hHSPCs were transplanted in NMDA or undamaged Nanog-GFP eyes. Quantification by FACS analysis of the reprogramming efficiency as the percentage of total GFP+ reprogrammed cells detected in the whole retinas (C) and quantification of GFP+ cells as percentage relative to the total number of transplanted DiD+ BIO-treated CD34+ hHSPCs counted in vertical sections, as for Figure S2B. Data are means ± s.e.m; n = 3.
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16. Figure S7
Analysis of Cell Fusion-Mediated Reprogramming after Transplantation of ESCs and RSCs, Related to Figure 2
(A) Representative retinal flat mounts where DiD-ESCs were injected 24 hr after PBS injection (control) or NMDA injection in Nanog-GFP-puro eyes. Twenty-four hours after ESCs injection, Nanog-GFP expression (green) is detected in ESC-neuron hybrids (red and green) in NMDA-damaged (NMDA) but not in non-treated (control) eyes.
(B) Confocal photomicrographs of vertical sections of undamaged (no NMDA: control) or NMDA-damaged Nanog-GFP-Puro eyes 24 hr after transplantation of DiD-labeled ESCs. Wnt-signaling after retinal damage was blocked with DKK1 injection (NMDA + DKK1), or activated with ESCs treatment with BIO or Wnt3a for 24 hr in culture before transplantation. Nuclei were counterstained with DAPI. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Scale bar: 20 μm.
(C) Quantification of double red+/green+ (DiD/YFP) hybrids detected in vertical sections of Nanog-GFP-puro undamaged or NMDA-damaged retinas treated or not with DKK1 and transplanted 24 hr later with untreated or Wnt3a-treated (Wnt3a) or BIO-treated (BIO) ESCs. Data are means ± s.e.m. (n = 90), counted in vertical sections (as for Figure S2B). ∗∗∗p <
(D) Histology of NMDA-damaged eyes from R26Y mice transplanted with BIO-treated ESCs and sacrificed one month after cell transplantation shows formation of YFP+ teratoma (bottom panel, green). Scale bar: 200 μm.
(E) Transplantation of DiD-labeled untreated RSPCs or of BIO-treated RSPCs (red) in NMDA-damaged Nanog-GFP eyes does not induce reactivation of the Nanog-GFP transgene (green). Nuclei were counterstained with DAPI. onl, outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. Scale bar: 20 μm.
(F) Quantification of YFP+ hybrids in R26Y eyes pre-treated or not with NMDA after injection of either untreated or BIO-treated (BIO) DiD-RSPCsCre (white bars), HSPCsCre (gray bars) or DiD-ESCsCre (black bars) counted in vertical sections (as for Figure S2B). Data are means ± s.e.m; n = 30.
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17. Figure S8
Characterization of In Vitro Cultured Reprogrammed Hybrids Obtained after Cell Fusion In Vivo, Related to Figure 2
(A) NMDA-damaged Nanog-GFP-Puro eyes were transplanted with BIO-treated (BIO) or untreated (No BIO) ESCsRFP. Undamaged eyes were transplanted with BIO-ESCsRFP as control. ESC-like clones emerged in culture after one month of puromycin selection only after transplantation of BIO-ESCsRFP.
(B) FACS analysis of RFP+/GFP+ hybrids isolated from NMDA-damaged Nanog-GFP retinas 24h after BIO-treated ESCsRFP transplantation.
(C) Representative alkaline phosphatase staining of reprogramed clones.
(D) A mean of 23 GFP+ clones were puromycin selected one month after transplantation of BIO-treated ESCs. Data are means ± s.e.m. (n = 3).
(E) Confocal micrographs of in vitro reprogrammed ES-like clones obtained from NMDA-damaged Nanog-GFP (green) retinas transplanted with BIO-treated ESCs and stained either with anti-Oct4 (Oct4, red) or anti-Nanog (Nanog, red) antibodies. Nuclei were counterstained with DAPI. Scale bar: 100 μm.
(F) FACS analysis of the DNA content in RFP+/GFP+ hybrids from NMDA-damaged retinas isolated 24h after BIO-treated ESCsRFP transplantation and stained with Hoechst. BIO-treated ESCsRFP and NMDA-damaged untransplanted retina was used as a control.
(G) Percentages of RFP+/GFP+ hybrids in G0/G1/S phase from 2n cells and in S/G2/M phase from 4n cells. Data are means ± s.e.m; n = 3.
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18. Figure S9 Characterization of ESC-Neuron Hybrids, Related to Figure 3
(A) Quantification of proliferating (BrdU+) YFP+ hybrids after transplantation of untreated (No BIO) or BIO-treated (BIO) HSPCsCre in NMDA-damaged R26Y eyes. Data are means ± s.e.m.; n = 30. ∗∗∗p <
(B) Quantification of mitotic (Ki-67+) and apoptotic (Annexin V+) YFP+ hybrids after transplantation of untreated (No BIO) or BIO-treated (BIO) ESCsCre in NMDA-damaged R26Y eyes. Data are means ± s.e.m.; n = 30. ∗∗∗p <
(C and D) Quantification of YFP+ hybrids positive to different markers, by immunostaining on sections counted (as for Figure S2B), at different times after BIO-treated (C) and non-treated (D) ESCsCre transplantation into NMDA-damaged eyes of R26Y mice. Data are means ± s.e.m; n = 30.
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19. Figure S10
Regeneration Analysis after Transplantation of HSPCs, Related to Figures 4 and 5
(A) Representative confocal image of flat-mounted NMDA-damaged retinas one month after transplantation of untreated HSPCsCre. Only a few YFP+ cells (green) were detected. Scale bars: 50 μm.
(B) Optic nerve harvested 24 hr after transplantation of HSPCsCre in NMDA-damaged R26Y eyes. Scale bars: 200 μm.
(C–G) FACS analysis showing percentages of RFP+/YFP+ hybrids also positive for CD45 (C, E) and Mac1 (D, F) staining 24 hr (C, D) and one month (E, F) after transplantation of HSPCsCre/RFP in NMDA-damaged R26Y eyes. The graph in G represents the mean values ± s.e.m. of three independent experiments.
(H) FACS analysis of YFP+ cells on the total retina 24h and 1 month after transplantation of BIO-treated HSPCsCre in NMDA-damages R26Y eyes. Data are means ± s.d. (n = 5).
(I) Real-time PCR analysis of the expression of the ganglion cell markers Calr and Brn3b in FACS sorted YFP+ hybrids obtained 1 month after transplantation of BIO-treated or untreated HSPCsCre in NMDA-damaged R26Y retinas. Values are represented as fold change with respect to the expression of the genes in a control retina. Data are means ± s.e.m.
(J) Confocal micrograph of flat-mounted NMDA-damaged R26Y retinas one month after transplantation of untreated HSPCsCre showing formation of pigmented YFP+ cells (green). Right panel show the merged image of YFP, DAPI and white field. Scale bar: 50 μm.
(K) Quantification of ganglion cell nuclei in NMDA-damaged flat mount retinas harvested one month after receiving Wnt3a treatment. All the nuclei excluding those of endothelial cells were counted in the ganglion cell layer along the nasotemporal (left) and dorsoventral (right) axes, and plotted as cells/mm2. A total of 80 different images composing the whole retina were counted for each sample. Data are means ± s.e.m. (n = 3).
(L) Total cells in the ganglion cell layer, excluding endothelial cells, were counted along the nasotemporal (left) and dorsoventral (right) axes and plotted as density maps, as indicated in the color key. ON, optic nerve.
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20. Figure S11
Analysis of Hybrids Formed after Mobilization of BM Cells in Damaged Eyes of Chimeric Mice, Related to Figure 7
(A) RFP+/YFP+ hybrids in flat-mounted retinas from chimeric R26Y mice after NMDA damage.
(B) Expression of Sca1, Chat, SMI-32 and GS proteins (red) evaluated in YFP hybrids (green) obtained after fusion of endogenously recruited BMCre cells with NMDA-damaged R26Y retinal cells. Arrows indicate double-positive hybrids.
(C) BIO injection in NMDA-damaged Nestin-Cre eyes leads to the expression of Nanog proteins (red) in YFP hybrids (green) obtained after fusion of endogenously recruited BMR26Y cells. Scale bar: 50 μm.
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