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Clonogenic Neoblasts Are Pluripotent Adult Stem Cells That Underlie Planarian Regeneration

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Clonogenic Neoblasts Are Pluripotent Adult Stem Cells That Underlie Planarian Regeneration

Daniel E Wagner et al. Science.

Abstract

Pluripotent cells in the embryo can generate all cell types, but lineage-restricted cells are generally thought to replenish adult tissues. Planarians are flatworms and regenerate from tiny body fragments, a process requiring a population of proliferating cells (neoblasts). Whether regeneration is accomplished by pluripotent cells or by the collective activity of multiple lineage-restricted cell types is unknown. We used ionizing radiation and single-cell transplantation to identify neoblasts that can form large descendant-cell colonies in vivo. These clonogenic neoblasts (cNeoblasts) produce cells that differentiate into neuronal, intestinal, and other known postmitotic cell types and are distributed throughout the body. Single transplanted cNeoblasts restored regeneration in lethally irradiated hosts. We conclude that broadly distributed, adult pluripotent stem cells underlie the remarkable regenerative abilities of planarians.

Figures

Figure 1
Figure 1. Expanding colonies are generated from isolated smedwi-1+ cells following irradiation
(A–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 1,750-rad treatment show clusters (arrowheads) of smedwi-1+ cells (individual purple dots). (C) Histogram of cluster frequencies following 1,750 rads. (D) Clusters observed by smedwi-1 ISH 7 days post-1,750 rads displayed in a scatterplot. phx, pharynx. (E–F) Animals fixed in a timecourse after 1,750-rad treatment analyzed by smedwi-1 fluorescence in situ hybridization (FISH). (F) Mean cluster frequency (#clusters/worm) and size (#smedwi-1+ cells/cluster) are plotted. Error bars, standard deviation (n=17–22 animals/timepoint). (G) IF (BrdU) and FISH (smedwi-1). 234/234 BrdU+ cells (8-hour BrdU-pulse in seven-day-irradiated worms) were smedwi-1+. (H) IF (SMEDWI-1) and FISH (smedwi-1); 12/12 colonies contained SMEDWI-1+; smedwi-1 cells (arrowheads) 7 days post-1,750 rads. (I) IF (BrdU) and FISH (smedwi-1). 31/31 colonies (with BrdU pulse days 7–11 post-1,750 rads) contained BrdU+; smedwi-1 cells. Scale bars, 200µm (A–B), 20µm (E, G–I).
Figure 2
Figure 2. Clonogenic neoblasts display broad differentiation capacity
(A–C) Triple-labeling of individual colonies 22 days after irradiation. Shown are projections through optical sections from irradiated animals. Left, tiled images (images from overlapping regions assembled) of representative animals with individual colonies are shown (anterior, up). Circles indicate approximate location of region imaged at high magnification for middle panels; 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 with arrowheads. See also Figure S12. (D) IF (BrdU) and double FISH (Smed-chat; Smed-mat) worms with BrdU-pulse days 14–18 after irradiation. Single colonies (n=7) contained both BrdU+; chat+ (neuronal) and BrdU+; mat+ (intestinal) descendants. Boxes indicate zoomed regions. (E) Scatterplots showing locations of individual colonies producing differentiated cell types (see also Figure S13). Colony cell differentiation was assessed by labeling with SMEDWI-1 (circles) or BrdU (diamonds). Scale bars, (A–C) left, 100µm; middle 20µm; right 5µm; (D) top image, 20µm; others 5µm.
Figure 3
Figure 3. Small numbers of cNeoblasts can restore tissue turnover and regenerative ability
(A–B) Animals were irradiated at different doses. Some of these animals were fixed 7 days post-irradiation (1,000 rads, 25 animals; between 1,125 and 1,875 rads, >38 animals/dose; for 6,000 rads, 26 animals) and labeled by smedwi-1+ ISH. (A) Representative smedwi-1+ ISH images. Anterior, left. (B) Colony numbers/worm are plotted (each dot represents one animal). (C) The percentage of animals with restored regeneration following irradiation (≥98 worms/dose were examined; animals were from the same irradiated cohort as in A–B). Data indicate 3 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 1,250 rads. (E) Heads regenerated after irradiation contained differentiated neuronal (chat+), intestinal (mat-1+), and muscle (mhc-1+) cells (41/41 worms, 1,250 rads; 15/15 worms, 1,500 rads). SMEDWI-1+ cells were also restored (n=9/9 worms, 1,250 rads). Dotted lines, approximate amputation plane. Arrowheads, photoreceptors. Scale bars, 200µm (A), 20µm (D–E).
Figure 4
Figure 4. Single transplanted cNeoblasts display properties of clonal growth and multipotency
Irradiation-sensitive cells (polygonal gate) were identified by Hoechst 333342 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 10µm in diameter with processes (arrowhead) (C). (D) Individual cells were loaded into needles (one needle used per injection) and transplanted into the medial, post-pharyngeal, parenchymal space of hosts. (E–F) FISH (smedwi-1) of a host immediately after transplantation. Anterior, up. Ventral surface shown. Zero (n=40/60) or one (n=20/60) smedwi-1+ cells were observed in all cases, with expected size and morphology. (F) is a zoomed image of (E). (G) Colony formation 9 days after irradiation, 6 days after transplant. Anterior, up. Ventral surface shown. Colonies of smedwi-1+ cells (arrowhead) appeared in transplant recipients (n=23/100) but not in untreated animals (n=5). (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); example differentiating cells from displayed colony are shown. Scale bars, 10µm (C), 50µm (E), 5µm (F) and zoomed images in (H), 20µm (H), 200µm (G).
Figure 5
Figure 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 regression site (dotted line) after seven weeks and fully regenerated after eight weeks. Anterior, up. Dorsal surface shown. (B) Representative images of rescue strains undergoing regeneration following amputation. Blastemas formed at approximate amputation plane (dotted line). Intestine (labeled with red food coloring) and photoreceptors (arrowheads) were observed in blastemas after 12 days of regeneration. Anterior, up. Dorsal surface shown. Scale bars, 1mm (A), 500µm (B).
Figure 6
Figure 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 line) and allowed to regenerate twice. (B) PCR-RFLP analysis of rescued strains. Locus 00310 was cut by HpaI in sexual animals (S) but not asexual animals (A) or the rescued strains (1, 2, 3). Locus 00463 was cut by ScaI in asexual animals and the rescued strains, but not in sexual animals. (C) Haplotype sequencing. Stacked histogram representing 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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