Establishment of a new method to isolate viable x-ray-sensitive cells from planarian by fluorescence-activated cell sorting

Abstract

Planarians show outstanding regenerative ability due to the proliferation of neoblasts. Hence the method to isolate planarian neoblasts is important to understand the regeneration process. In our previous study, we reported a method to isolate planarian neoblasts of Dugesia japonica using fluorescence-activated cell sorting (FACS). However, we have not yet succeeded in cultivating these cells even under in vivo conditions after transplantation into x-ray-irradiated planarians. This suggests that dissociated cells might enter apoptotic or necrotic states in the process of fluorescent dye staining and sorting. Here, we developed a new method to isolate viable neoblasts, which can proliferate in the x-ray-irradiated planarians. First, the toxicity of various fluorescence dyes was investigated. All nuclear fluorescent dyes such as Hoechst 33342, DRAQ5, and DyeCycle, showed, more or less, toxicity to mammalian culture cells. In contrast, cytoplasmic fluorescent dye for live cells, calcein AM, was less toxic on these cells. Next, we stained the dissociated planarian cells with only calcein AM, and then collected the x-ray-sensitive fraction. Although the purity of neoblasts was slightly lower than that of the original staining method (ca. 97% → ca. 89%), the sorted cells could actively proliferate when they were injected into x-ray-irradiated planarians. This simple staining and sorting method will provide new opportunities to isolate viable neoblasts and understand regenerating processes.

Keywords: calcein AM; fluorescence-activated cell sorting; neoblasts; planarian; transplantation.

FIGURE 1

Hoechst 33342 and DRAQ5, conventional reagents for neoblasts purification, are highly cytotoxic. (a, b) Growth curves of PC12 and EB5 cells without staining or stained with 32 μM Hoechst 33342, 0.5 μg/mL calcein AM, or both. (c, d) Growth curves for PC12 and EB5 cells without staining or stained with 5 μM DRAQ5, 0.4 μg/mL calcein AM, or both. Error bars indicate the mean ± SEM (n = 3).

FIGURE 2

Lower concentrations of Hoechst 33342 have less cytotoxicity. (a, b) Growth curves for PC12 and EB5 without staining or stained with 1, 3, 5, 10, or 32 μM Hoechst 33342. (c, d) Growth curves for PC12 and EB5 without staining or stained with 1, 3, or 5 μM DRAQ5. (e, f) Growth curves for PC12 and EB5 without staining or stained with 1, 3, 5, 10, or 32 μM DyeCycle. Error bars indicate the mean ± SEM (n = 3).

FIGURE 3

The x‐ray‐sensitive fraction was observed in the Hoechst‐low/FSC‐H‐high region. (a) Fluorescence‐activated cell sorting (FACS) plots for planarian cells stained with 1, 3, 5, or 10 μM Hoechst 33342 for 30 min and stained with 32 μM Hoechst 33342 for 120 min. The number of cells displayed is 10,000 cells in the plots for 1–10 μM Hoechst 33342 and 20,000 cells in the plot for 32 μM Hoechst 33342. (b) FACS plots for planarian cells stained with 1, 3, 5, or 10 μM Hoechst 33342 for 30 min after adjustment of FACS gain. The gain configuration for Hoechst 33342 was increased according to 32 μM Hoechst 33342 in (a). The number of cells displayed is 10,000 cells. Dashed box, x‐ray‐sensitive fraction; solid‐line box, Hoechst‐high region.

FIGURE 4

X‐ray‐sensitive cells isolated by staining with low concentrations of Hoechst 33342 consisted of nearly 90% X1 fraction cells. (a) A schematic of the experimental flow. (b) FACS plots of planarian x‐ray‐sensitive cells sorted by staining with 1, 3, 5, or 10 μM Hoechst 33342 for 30 min. Solid‐line boxes represent the X1 fraction obtained by the original staining method (Hayashi et al., 2006). (c) Percentage of X1 fraction cells among Hoechst 33342 and calcein AM double‐positive cells. Error bars indicate the mean ± SEM (n = 3).

FIGURE 5

X‐ray‐sensitive cells isolated by “the only calcein AM” staining method consisted of about 90% X1 fraction cells. (a) Fluorescence‐activated cell sorting (FACS) plots of planarian cells stained with 0.05 μg/mL calcein AM for 30 min. X_S, X‐ray‐sensitive small size; X_L, X‐ray‐sensitive large size. (b) FACS plots of planarian X_S and X_L fraction cells isolated by the original staining method (Hayashi et al., 2006). Solid‐line boxes represent the X1 fraction. (c) Percentage of X1 fraction cells among Hoechst 33342 and calcein AM double positive cells. Error bars indicate the mean ± SEM (n = 3).

FIGURE 6

X‐ray‐sensitive cells isolated by “the only calcein AM” staining method have the highest survival rate. (a) Immunostaining of DjPiwiA in x‐ray‐sensitive cells sorted by the original staining method, “the Hoechst low” staining method and “the only calcein AM” staining method. (i–iii) An enlarged image of the region in the dashed box in the merge image. Scale bars, 30 μm. (b) Percentage of DjPiwiA‐positive cells among Hoechst 33342 positive cells isolated by the original staining method, “the Hoechst low” staining method and “the only calcein AM” staining method. n = 10, respectively. *p < 0.05; NS, not significant by one‐way ANOVA followed by Tukey's multiple‐comparisons test. (c) The survival rate of cultured x‐ray‐sensitive cells isolated by three staining methods. Error bars indicate the mean ± SEM (n = 5). *p < 0.05; **p < 0.01; NS, not significant by one‐way ANOVA followed by Tukey's multiple‐comparisons test.

FIGURE 7

The transplanted cells isolated by “the only calcein AM” staining method propagated in x‐ray‐irradiated planarians. (a–h) Whole‐mount immunostaining of DjPiwiA in ventral view. (a) Intact planarian. (b–g) Representative examples of x‐ray‐irradiated planarians 2 weeks after the injection into the post‐pharyngeal area. The injected x‐ray‐sensitive cells were purified by “the only calcein AM” method. (h) X‐ray‐irradiated planarian without injection. (a’–h’) An enlarged image of the region in the dashed box in (a–h), respectively. Scale bars, 500 μm (a–h); 50 μm (a’–h’).

FIGURE 8

The transplanted cells isolated by “the only calcein AM” staining method maintain proliferative activity. (a–d) Whole‐mount immunostaining of DjPiwiA and pH 3 in ventral view. (a) Intact planarian. (b, c) Representative examples of x‐ray‐irradiated planarians 2 weeks after the injection. The injected x‐ray‐sensitive cells were isolated by “the only calcein AM” method. (b) Cells were injected into the post‐pharyngeal area. (c) Cells were injected into both the head area and post‐pharyngeal area. (d) X‐ray‐irradiated planarian without injection. (a’–d’) An enlarged image of the region in the dashed box in (a–d), respectively. Scale bars, 500 μm (a–d); 50 μm (a’–d’).