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Science 332 (6031): 811-816
Copyright © 2011 by the American Association for the Advancement of Science
Clonogenic Neoblasts Are Pluripotent Adult Stem Cells That Underlie Planarian Regeneration
Daniel E. Wagner1,*,
Irving E. Wang1,*, and
Peter W. Reddien1,
1 Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology (MIT), Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.
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Fig. 1. Expanding colonies are generated from isolated smedwi-1+ cells after irradiation. (A and B) Proliferating cells were detected by smedwi-1 expression using whole-mount in situ hybridization (ISH). Anterior, up; ventral surface shown. (B) Representative images 7 days after 1750-rad treatment show clusters (arrowheads) of smedwi-1+ cells (individual purple dots). (C) Histogram of cluster frequencies after 1750-rad treatment. The dashed red box indicates that the majority of animals contained either zero or one cluster. (D) Clusters observed by smedwi-1 ISH 7 days post–1750-rad treatment displayed in a scatter plot. phx, pharynx. (E and F) Animals fixed in a time course after 1750-rad treatment analyzed by smedwi-1 fluorescence in situ hybridization (FISH). d, days. (F) Mean cluster frequency (number of clusters per worm) and size (number of smedwi-1+ cells per cluster) are plotted. Error bars indicate SD (n = 17 to 22 animals per time point). (G) Immunofluorescence (IF) (BrdU) and FISH (smedwi-1). 234/234 BrdU+ cells (8-hour BrdU pulse in 7-day–irradiated worms) were smedwi-1 positive. (H) IF (SMEDWI-1) and FISH (smedwi-1); 12/12 colonies contained SMEDWI-1+; smedwi-1– cells (arrowheads) 7 days post–1750-rad treatment. (I) IF (BrdU) and FISH (smedwi-1). 31/31 colonies (with BrdU pulse days 7 to 11 post-1750 rad) contained BrdU+; smedwi-1– cells. Scale bars, 200 μm [(A) and (B)]; 20 μm [(E) and (G) to (I)].
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Fig. 2. Clonogenic neoblasts display broad differentiation capacity. (A to C) Triple-labeling of individual colonies 22 days after irradiation. Projections through optical sections from irradiated animals are shown here. (Left) Tiled images (images from overlapping regions assembled) of representative animals with individual colonies (anterior, up). Dotted circles indicate the approximate location of regions imaged at high magnification (middle panels); the middle images are optical sections with anterior to the right. Example differentiating cells from individual colonies labeled by IF (SMEDWI-1) and double FISH for gata4/5/6 and chat (A), gata4/5/6 and AGAT-1 (B), or AGAT-1 and chat (C) are shown. Proportions of colonies displaying multiple differentiating cell types are indicated. Roman numerals indicate double-positive cells, with individual channels shown in columns to the right. Additional double-positive cells are indicated by arrowheads (see also fig. S12). (D) IF (BrdU) and double FISH (Smed-chat; Smed-mat) worms with BrdU-pulse days 14 to 18 after irradiation. Single colonies (n = 7/7 colonies) contained both BrdU+; chat+ (neuronal) and BrdU+; mat+ (intestinal) descendants. Boxes indicate zoomed-in regions. (E) Scatter plots showing locations of individual colonies producing differentiated cell types (see also fig. S13). Colony cell differentiation was assessed by labeling with SMEDWI-1 (circles) or BrdU (diamonds). Scale bars, (A to C) left, 100 μm; middle 20 μm; right 5 μm; (D) top image, 20 μm; others, 5 μm.
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Fig. 3. Small numbers of cNeoblasts can restore tissue turnover and regenerative ability. (A and B) Animals were irradiated at different doses. Some of these animals were fixed 7 days post-irradiation (1000 rad, 25 animals; between 1125 and 1875 rad, >38 animals per dose; for 6000 rad, 26 animals) and labeled by smedwi-1+ ISH. (A) Representative smedwi-1+ ISH images. Anterior, left. (B) Colony numbers per worm are plotted (each dot represents one animal). (C) Percentage of animals with restored regeneration after irradiation is shown [ 98 worms per dose were examined; animals were from the same irradiated cohort as in (A) and (B)]. Data indicate that three or more cNeoblasts were sufficient to restore regeneration (see also table S1). (D) Normal head regeneration in 97/99 worms amputated 39 or 40 days after 1250-rad treatment. Arrowheads denote photoreceptors. (E) Heads regenerated after irradiation contained differentiated neuronal (chat+), intestinal (mat-1+), and muscle (mhc-1+) cells (41/41 worms, 1250 rad; 15/15 worms, 1500 rad). SMEDWI-1+ cells were also restored (n = 9/9 worms, 1250 rad). Dotted lines denote the approximate amputation plane. Scale bars, 200 μm (A); 20 μm [(D) to (E)].
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Fig. 4. Single transplanted cNeoblasts display properties of clonal growth and multipotency. Irradiation-sensitive cells (polygonal gate) were identified by Hoechst 33342 labeling (A) and back-gated to set the X1(FS) gate (oval) based on size (FS) and complexity (SS) parameters (B). The X1(FS) fraction is heterogeneous and contains some cells approximately 10 μm in diameter with processes (arrowhead) (C). (D) Individual cells were loaded into needles (one needle used per injection) and transplanted into the medial, postpharyngeal, parenchymal space of hosts. (E and F) FISH (smedwi-1) of a host immediately after transplantation. Anterior, up. The ventral surface is shown. Zero (n = 40/60 animals) or one (n = 20/60 animals) smedwi-1+ cells were observed in all cases, with expected size and morphology. (F) is a zoomed-in image of (E). (G) Colony formation 9 days after irradiation, 6 days after transplant. Anterior, up. The ventral surface is shown. Colonies of smedwi-1+ cells (arrowhead) appeared in transplant recipients (n = 23/100 animals) but not in untreated animals (n = 0/5 animals). px, pharynx. (H) IF (SMEDWI-1) and double FISH (Smed-gata4/5/6; Smed-chat) 33 days after irradiation, 30 days after transplant. Single colonies were observed (n = 4/17 animals); example differentiating cells from displayed colony are shown. Scale bars, 10 μm (C); 50 μm (E); 5 μm (F) and zoomed images in (H); 20 μm (H); 200 μm (G).
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Fig. 5. Restoration of regeneration in lethally irradiated hosts by single transplanted cNeoblasts. (A) Representative images of transplant hosts. Tissue regression (asterisks) began anterior to photoreceptors (arrowheads) and progressed from anterior to posterior (px, pharynges). Rescued animals developed blastemas (arrow) at the regression site (red dotted line) after 7 weeks and fully regenerated after 8 weeks. Anterior, up. The dorsal surface is shown. (B) Representative images of rescue strains undergoing regeneration after amputation. Blastemas formed at approximate amputation plane (red dotted line). Intestine (labeled with red food coloring) and photoreceptors (arrowheads) were observed in blastemas after 12 days of regeneration. Anterior, up. The dorsal surface is shown. Scale bars, 1 mm (A); 500 μm (B).
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Fig. 6. Genotype conversion by single transplanted cNeoblasts. (A) Schematic showing replacement of host tissue by transplanted donor cells (blue); animals for genotyping were amputated (dotted lines) and allowed to regenerate twice. (B) PCR-RFLP analysis of rescued strains. Locus 00310 was cut by HpaI in asexual animals (A) and the rescued strains (1, 2, 3), but not in sexual animals (S). Locus 00463 was cut by ScaI in sexual animals, but not in asexual animals or the rescued strains. (C) Haplotype sequencing (22). Stacked histogram representing the number of sequencing reads from each locus for each strain. Bars extend left for number of reads corresponding to the asexual haplotype and right for number of reads corresponding to the sexual haplotype. Bar absence indicates no reads.
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