Candidate stem cell isolation and transplantation in Hexacorallia

Abstract

Stem cells are the foundation for cell therapy due to their ability to self-renew, differentiate into other cell types, and persist throughout the life of an organism. Stem cell isolation and transplantation have not yet been established in Hexacorallia, a cnidarian subclass containing stony corals and sea anemones. Here, we demonstrate that candidate stem cells in the hexacorallian Nematostella vectensis can be transplanted into adult animals. These cells exhibited the hallmarks of stem cell functional properties; they integrated into recipients' tissues and rescued them from lethal doses of chemotherapy. Additionally, these cells proliferated and survived serial transplantations. Notably, we showed that this cellular subpopulation can be enriched by sorting using species-non-specific cell markers and that similar subpopulations of cells can be isolated from other hexacorallians, including stony corals. This research establishes the basis for studying stem cell biology on a functional level in Hexacorallia.

Keywords: CP: Stem cell research; Hexacorallia; Nematostella vectensis; cell therapy; corals; stem cells; transplantation.

Graphical abstract

Figure 1

Cell transplantation in Nematostella vectensis (A) In the experimental setting, recipient wild-type (WT) N. vectensis was treated with mitomycin C (MITC) 3 days before the transplantation. Donor animals expressing the mCherry reporter under the regulation of the housekeeping TBP promoter (transgenic TBP::mCherry) were then dissociated, and single-cell suspensions of approximately 100,000 donor cells were transplanted by microinjection into each MITC-treated recipient animal. Donor-derived mCherry signal in the recipients was periodically assessed by confocal/fluorescent microscopy for up to 2 months. At the end of the experiment, the donor-derived mCherry signal was assayed by FACS and PCR. (B) mCherry signal in recipient animal mesentery area 7 days after transplantation of donor cells. Bright-field image (left), fluorescent mCherry image (center), merged image (right). Scale bar, 100 μm. (C) Representative experiment showing the percentage of recipient animals with mCherry signal over time. Ten transplanted and 10 non-transplanted animals that had been MITC treated were monitored for mCherry on the days indicated. (D and E) Frequency of mCherry⁺ cells 40 days after transplantation. Eleven transplanted animals, 10 non-transplanted animals, 1 donor animal (triplicate, positive control), and 1 WT animal (triplicate, negative control) were dissociated to generate single-cell suspensions. mCherry signal was assessed by flow cytometry. (D) Representative results for a single animal in each group are shown. The red gate shows the positive cutoff values against non-specific basal autofluorescence of the native protein NvFP-7R (PE-Cy7).^, (E) Quantitative results of all the animals in the experiment (median, 25th–75th percentiles, and minimum–maximum values shown, ^∗∗∗∗p ≤ 0.0001, unpaired t test). (F) mCherry DNA in transplanted recipients. DNA was extracted from an individual donor/positive control (n = 1), transplanted (n = 12), and non-transplanted (n = 9) animals at the end of the experiment. The presence of mCherry DNA was assessed by PCR amplification of a 643-bp mCherry-specific DNA sequence. Co-amplification of a 381-bp HKG4 sequence found in all the genomes served as an internal positive control (^∗, individuals scored positive). Ladder on the left, PCRBIO ladder II (250 bp−10 kb; C#PB40.12-01).

Figure 2

Recipient rescue and transplanted cell longevity (A and B) For survival assays, non-transplanted animals were treated with a lethal dose of MITC (75 μM) and 3 days later injected with donor cells. Their viability was scored weekly. (A) Survival curve of one representative experiment of 11 transplanted and 10 non-transplanted animals. (B) Four rescue experiments were repeated to yield 34 animals in the transplanted group and 32 in the non-transplanted group. The fraction of surviving animals 4–8 weeks after transplantation was calculated (^∗p = 0.0103, Fisher’s exact test). (C and D) For longevity, WT recipient animals were treated with a sublethal dose of MITC and then injected with TBP::mCherry donor cells. T 6–8 weeks after the transplantation, the recipient animals were dissociated, and their mCherry⁺ cells were collected by FACS. A total of 40,000 of these were then transplanted again into sublethally MITC-treated recipient animals. Twelve transplanted animals and 15 non-transplanted animals (see Figure S3A) were incubated and scored weekly for mCherry signal for 6 weeks (C), a representative experiment. (C) A representation of the percentage of animals expressing the mCherry signal over time after the longevity transplantation experiment (n = 6 in each group). (D) Confocal microscope images illustrating the mCherry signal in a transplanted animal, 31 days after serial transplantation (D, top) and a non-transplanted animal under the same conditions (D, bottom; scale bar, 100 μm).

Figure 3

A subpopulation of cells expressing high-ALDH activity with low granularity is enriched for candidate stem cells (A–E) Recipient WT animals were treated with a sublethal dose of MITC 3 days before transplantation. mCherry-expressing donor animals were dissociated, and the cells were then labeled with ALDEFLUOR. FACS was used to isolate an ALDH-high/low granularity (low side-scatter) cell subpopulation and a control ALDH-low/high granularity cell subpopulation. Approximately 10,000 cells of one subpopulation or the other were transplanted into each recipient. (A) Cell morphology in sorted donor cell subpopulations (ALDH-high, ALDH-low) was observed by confocal microscopy (scale bar, 10 μm). (B) Donor cells in recipients 20 days after transplantation of ALDH-high cells (right) or ALDH-low cells (left) as observed by confocal microscopy (scale bar, 100 μm/50 μm for the upper right). (C) Fraction of mCherry⁺ recipient animals over time. Recipients were pre-treated with MITC and transplanted either with ALDH-high (n = 10), ALDH-low (n = 10), or non-transplanted (n = 9) accordingly. Recipient animals were scored as mCherry⁺ or mCherry⁻ by fluorescent microscopy on the indicated days following transplantation. The representative experiment is shown. (D) Abundance of donor-derived cells in recipients after 2 months. The recipient animals were dissociated, and the fraction of mCherry⁺ donor-derived cells was measured by flow cytometry. n = 10 for each group (median, 25th–75th percentiles, and minimum–maximum values are shown; ^∗∗∗p = 0.0002; ^∗∗∗∗p < 0.0001; ordinary one-way ANOVA). (E) Recipient survival following transplantation. Survival in the indicated groups was measured 55 days after transplantation. Triplicate ALDH-high/-low experiment yielded 40 animals in the ALDH-high transplanted group, 30 animals in the ALDH-low transplanted group, and 29 animals in the non-transplanted group (^∗∗∗p = 0.0002; ^∗∗p = 0.0012; ns, p = 0.6010; Fisher’s exact test was performed on each comparison individually). (F) Proliferation of donor-derived (mCherry⁺) cells after transplantation. At 31 days after transplantation, recipient animals were incubated with EdU for 4.5 h. (Left) Comparing the proliferation of donor-derived (mCherry⁺; purple) and host (mCherry⁻; blue) cells in three different animals transplanted with ALDH-high cell population and measured in quadruplicate (median, 25th–75th percentiles, and minimum–maximum values are shown; ^∗∗∗∗p < 0.0001; two-way ANOVA). (Right) Comparing the proliferation of donor-derived (mCherry⁺) ALDH-high and ALDH-low cells in recipient animals (n = 3 in both groups) (median, 25th–75th percentiles, and minimum to maximum values are shown; p = 0.0032; unpaired t test).

Figure 4

Differentiation of candidate stem cells after transplantation (A–D) Approximately 10,000 donor-derived ALDH-high, mCherry⁺ cells (A, left) were injected into MITC-treated WT recipients. Two months after transplantation, the recipients were dissociated, and donor-derived mCherry⁺ cells were sorted by FACS (A, center). Total recipient cells are also shown (A, right; scale bar, 10 μm). Arrows indicate apparent nematocysts (A and B). (B) Cell field images from the three cell populations were provided to a machine learning program that automatically identified cell outlines (in red; scale bar, 10 μm). (C and D) The cell outlines were used by the machine learning algorithm to calculate cell areas (C) (n = 12 images in mCherry⁺ and total, n = 11 images for ALDH-high; Brown-Forsythe and Welch ANOVA tests; ns, p = 0.0894; ^∗∗∗∗p < 0.0001) and circularity. (D) Two-dimensional analysis of cell area vs. circularity comparing the analyzed cells of mCherry⁺ vs. the transplanted ALDH-high (left) and vs. total cell population (right). (E) Cell differentiation is shown by flow cytometry analysis. Cell morphology is indicated by FSC (represents size) vs. SSC (represents granularity and cell complexity) on a logarithmic scale. From left to right: ALDH-high cell population before transplantation, donor-derived cell population (mCherry⁺) 2 months after ALDH-high cell transplantation, and total cell population with no transplantation applied.

Figure 5

The mesenteries as a putative stem cell niche and the location for ALDH-high cell population (A) To locate the ALDH-high cell population, we injected ALDEFLUOR green (GFP channel) into live transgenic N. vectensis (TBP::mCherry; mCherry channel). Top: control transgenic N. vectensis expressing the mCherry reporter under the regulation of the housekeeping TBP promoter (transgenic TBP::mCherry; red) without treatment. Bottom: Another transgenic TBP::mCherry (red) 30 min after injection of the ALDH marker (ALDEFLUOR; green). Fluorescent GFP image (left), fluorescent mCherry image (center), merged image (right) (scale bar, 1 mm). (B–D) Cell transplantation from different N. vectensis body parts. (B) Confocal picture showing donor-derived (mCherry⁺) cells in a tentacle after the transplantation of cells from the mesenteries (scale bar, 50 μm). (C) Flow cytometry analysis of donor-derived cells (mCherry⁺) 41 days after cell transplantation of different body parts, including pharynx, mesenteries, and physa. n = 6 for pharynx transplanted group, n = 5 for physa transplanted group, and n = 12 for the mesenteries transplanted group (median, 25th–75th percentiles, and minimum–maximum values shown. ^∗∗p = 0.0073; ^∗p = 0.0173; ns, p = 0.9862; ordinary one-way ANOVA). (D) A volcano plot representing the difference between cells from mesenteries (n = 4) and the donor-derived (mCherry⁺) cells from animals transplanted with cells isolated from mesenteries (n = 6) 41 days after the transplantation. Differentially expressed genes with absolute log2 fold change (FC) >1 and adjusted p < 0.05 are shown in green. Associated stem cell markers and cell-type/tissue-specific markers are highlighted.