Isolation and characterization of neural stem/progenitor cells in the subventricular zone of the naked mole-rat brain

Background The naked mole-rat (NMR) is the longest-lived rodent with a maximum lifespan of more than 37 years and shows a negligible senescence phenotype, suggesting that tissue stem cells of NMRs are highly capable of maintaining homeostasis. However, the properties of NMR tissue stem cells, including neural stem cells (NSCs), are largely unclear. Methods Neural stem/progenitor cells (NS/PCs) were isolated from the subventricular zone of the neonate NMR brain (NMR-NS/PCs) and cultured in neurosphere and adherent culture conditions. Expression of NSC markers and markers of neurons, astrocytes, and oligodendrocytes was analyzed by immunocytochemistry. In adherent culture conditions, the proliferation rate and cell cycle of NMR-NS/PCs were assessed and compared with those of NS/PCs from mice (mouse-NS/PCs). The DNA damage response to γ-irradiation was analyzed by immunocytochemistry and reverse transcription-quantitative PCR. Results NMR-NS/PCs expressed several NSC markers and differentiated into neurons, astrocytes, and oligodendrocytes. NMR-NS/PCs proliferated markedly slower than mouse-NS/PCs, and a higher percentage of NMR-NS/PCs than mouse-NS/PCs was in G0/G1 phase. Notably, upon γ-irradiation, NMR-NS/PCs exhibited a faster initiation of the DNA damage response and were less prone to dying than mouse-NS/PCs. Conclusions NMR-NS/PCs were successfully isolated and cultured. The slow proliferation of NMR-NS/PCs and their resistance to DNA damage may help to prevent stem cell exhaustion in the brain during the long lifespan of NMRs. Our findings provide novel insights into the mechanism underlying delayed aging of NMRs. Further analysis of NMR tissue stem cells may lead to the development of new strategies that can prevent aging in humans. Supplementary Information The online version contains supplementary material available at 10.1186/s41232-021-00182-7.


Background
Naked mole-rats (NMRs) are the longest-lived rodent species and live underground in northeastern Africa [1]. The maximum lifespan of NMRs exceeds 37 years despite their body size being similar to that of laboratory mice [2]. Notably, Ruby et al. reported that NMRs do not show an increase in mortality with age, an important indicator of organismal aging [3]. Moreover, NMRs exhibit extraordinary resistance to cancer; spontaneous carcinogenesis was seldom observed in more than 2000 necropsies of captive NMR colonies [4][5][6]. These unusual characteristics make NMRs an attractive animal model for obtaining clues to prevent aging and cancer in humans.
Recently, several studies proposed potential mechanisms underlying the longevity and cancer resistance of NMRs involving protein stability [7], activity of nuclear factor, erythroid 2-like 2 (NRF2) signaling [8], translational fidelity [9], production of high molecular mass hyaluronic acid [10], the unique cellular response to reprogramming/oncogenic stress [11,12], and the retrotransposon [13]. NMRs also have a high DNA repair capacity. Tian et al. reported that compared with shortlived rodent species, fibroblasts from various long-lived rodent species including NMRs have a high DNA double-strand break (DSB) repair capacity and high activity of Sirtuin 6, which acts as a DSB sensor [14,15]. In addition to their high DSB repair capacity, NMRs have high DNA excision repair activity [16].
Stem cells can self-renew in an undifferentiated state and differentiate into at least one specialized cell type. We and other groups previously generated induced pluripotent stem (iPS) cells from NMR fibroblasts and showed that these cells exhibit marked tumor resistance when transplanted into immunodeficient mice [11,12,17]. Tissue stem cells, which exist in various locations such as the bone marrow, intestine, muscle, and brain, play important roles in maintaining body homeostasis and repairing tissues [18]. Depletion or dysfunction of tissue stem cells is one of the major causes of aging and cancer [19]. Neural stem cells (NSCs) are tissue stem cells located in the central nervous system and give rise to neurons, astrocytes, and oligodendrocytes [20]. In the postnatal and adult rodent brain, most NSCs are located in the subventricular zone (SVZ) and the subgranular zone (SGZ) [21]. In these niches, most NSCs are maintained in a quiescent state, which is a reversible state of cell cycle arrest, and a small population of NSCs is activated by various stimuli and generates neurons [22]. The balance between stem cell quiescence and activation is crucial for long-term maintenance of the stem cell pool and the neurogenic capacity of the brain during aging [23]. In addition, recent reports suggested that highly malignant brain tumors originate from neural stem/ progenitor cells (NS/PCs) [24,25]. Therefore, NSCs in NMRs may be highly capable of maintaining cellular homeostasis, which may be related to resistance to aging and cancer.
Adult neurogenesis occurs in the SVZ and SGZ of the NMR brain [26][27][28]. However, isolation and culture of NMR NSCs have not been previously reported. Therefore, the properties of these cells are largely unknown. In this study, we isolated NS/PC populations from the SVZ of NMR neonates (NMR-NS/PCs) and characterized their basic properties and DNA repair capacity.

Animals
The Ethics Committees of Kumamoto University approved all procedures (approval no. A30-043 and A2020-042). The procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (United States National Institutes of Health). NMRs were maintained at Kumamoto University. C57BL/6N mice were purchased from Japan SLC, Inc.

Primary culture of NS/PCs
To obtain NMR-NS/PCs and NS/PCs from mice (mouse-NS/PCs), SVZs of neonatal NMRs and C57BL/ 6N mice at postnatal days 1-2 were isolated, as previously described [29]. The isolated SVZs were washed with ice-cold phosphate-buffered saline (PBS; Nacalai Tesque) containing 0.6% glucose, 1% penicillin/streptomycin (FUJIFILM Wako), and amphotericin B (FUJI-FILM Wako). The SVZs were dissociated in growth medium (described below) containing 1.3 mg/ml trypsin (Sigma-Aldrich), 0.7 mg/ml hyaluronidase (Sigma-Aldrich), and 0.1 mg/ml DNase I (Sigma-Aldrich) at 32°C for 10 min. To stop the enzymatic reaction, an equivalent volume of a trypsin inhibitor solution (growth medium containing 2 mg/ml trypsin inhibitor and 0.1 mg/ml DNase I) was added, and the samples were dissociated by pipetting. Cells from each neonate were collected by centrifugation, washed with growth medium, and seeded in a single well of a 6-well plate in growth medium, which comprised Dulbecco's modified Eagle medium: Nutrient Mixture F-12 (Sigma-Aldrich) supplemented with 2% B27 Supplement minus Vitamin A (Gibco), 0.5% penicillin/streptomycin, 2 mM Lglutamine (Nacalai Tesque or FUJIFILM Wako), 200 ng/ ml heparan sulfate sodium salt from bovine kidney (Sigma-Aldrich), and 20 ng/ml of both basic fibroblast growth factor (bFGF; Pepro Tech EC) and epidermal growth factor (EGF; Pepro Tech EC). Half of the medium was replaced with fresh medium every 5 days. When spheres exceeded 100 μm in diameter, they were passaged using 0.05% trypsin-EDTA and 2 mg/ml trypsin inhibitor containing 0.1 mg/ml DNase I, and plated at a density of 1.5 × 10 6 cells per plate in 100 mm ultralow attachment dishes (Corning). For adherent culture, cells were plated at a density of 1.5 × 10 6 cells per plate in poly-L-ornithine/laminin-coated 100 mm plates. For differentiation, neurospheres were plated onto poly-Lornithine/fibronectin-coated coverslips in a 48-well plate, and cells in adherent culture conditions were plated at a density of 4 × 10 4 cells per well onto poly-Lornithine/fibronectin-coated coverslips in a 48-well plate and allowed to differentiate without growth factors for 10 days.

Cell growth analysis
NMR-and mouse-NS/PCs were seeded at a density of 8 × 10 4 cells per well in a 24-well plate. Seeded cells were trypsinized and counted every 3 days using a Coulter Counter (Beckman Coulter). The population doubling time was estimated from the slope of the growth curve.

Cell cycle analysis
Cells in the logarithmic growth phase were collected and fixed with 70% ethanol at 4°C for 2 h. After two washes with PBS, cells were incubated with RNase A (QIAGEN, final concentration of 0.25 mg/ml) at 37°C for 45 min and then stained with propidium iodide (PI; FUJIFILM Wako, final concentration of 50 μg/ml) at 4°C for 30 min in the dark. Flow cytometry was performed using a FACSVerse flow cytometer (BD Biosciences). Data were analyzed using FlowJo 10 software (BD Biosciences).

γ-Irradiation and cell death analysis
Cells were seeded into 24-well plates at a density of 1.6 × 10 5 cells per well. At 24 h after seeding, cells were exposed to 10 Gy of γ-radiation. To detect cell death, cells were stained with Hoechst 33342 (Sigma; 1 mg/ml; 1: 1000 in growth medium) for 10 min at 32°C and then stained with PI (10 mg/ml; 1:1000 in growth medium) for 10 min at 32°C. Images were captured using a BZ-X 710 fluorescence microscope (Keyence) and positively stained cells were counted using a BZ-X Image Analyzer (Keyence). All cells in 12 randomly selected microscope fields (at least 10,000 cells) per cell line were analyzed for PI-and Hoechst-positive nuclei. PI-and Hoechstdouble-positive cells were counted as dead cells.

Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted using TRIzol (Thermo Fisher Scientific) according to the manufacturer's protocol. Genomic DNA was removed using gDNA Eliminator spin columns (Qiagen). RNA was eluted from the columns using 30 μl of RNase-free water and quantified using a NanoDrop spectrophotometer (Thermo Scientific). cDNA was synthesized with ReverTra Ace qPCR RT Master Mix (TOYOBO) using 400 ng of total RNA input. RT-qPCR assays were set up in triplicate using PowerUp SYBR Green Master Mix (Invitrogen) and run on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). Primer sequences are listed in Supplementary  Table 1.

Statistical analysis
GraphPad Prism was used for statistical analysis. Data are presented as the mean ± standard deviation (SD). Data were analyzed using a two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test or a one-way ANOVA followed by Dunnett's multiple comparison test. The unpaired t-test was used to compare two groups.

Isolation and characterization of NMR-NS/PCs
We isolated NS/PCs from early neonatal (postnatal days 1-2, P1-2) NMRs (Fig. 1a). Immunohistochemistry showed that the SVZ of the neonatal NMR brain contained many cells positive for the NSC markers SOX2, NES, and MSI1, as well as a small population of BrdU (a proliferation marker)-positive cells (Fig. 1b, c, Supplementary Fig. 1a). In the NMR SVZ, there were many BrdU-positive cells not only in the lateral wall but also in the medial wall ( Supplementary Fig. 1b). Upon immunohistochemical staining of the SVZ for SOX2, strongly positive cells were observed in the area near the wall and weakly positive cells were observed in the area distant from the wall (Supplementary Fig. 1c). Although the amino acid sequence of NES varies among species, the amino acid homology of the antigen site between the human and NMR proteins is 73.3%, and the antibody showed cross-reactivity ( Supplementary Fig. 1d). To isolate and culture NS/PCs, the SVZ of NMRs was microdissected as previously described in mice (Fig. 1d) [29]. The dissected SVZ was dissociated by gentle pipetting after treatment with trypsin and hyaluronidase, suspended in NSC culture medium supplemented with B27, bFGF, and EGF, as previously used in other species [31], and cultured in neurosphere culture conditions. The NMR is a unique heterothermic animal with a low body temperature and its cells are vulnerable to culture in normoxia [17,32]; therefore, the cells were cultured at 32°C and in 3% O 2 . Neurospheres formed after 15 days of cultivation (Fig. 1e). To eliminate adherent differentiated cells, neurospheres were cultured in a cell culture flask and passaged three times. Dissociated neurospheres could grow on poly-L-ornithine/laminin-coated dishes (adherent culture, Fig. 1e). These cells could be cultured up to at least passage six in both neurosphere and adherent culture conditions. Immunocytochemistry showed that most cells were positive for the NSC markers SOX2, NES, and MSI1 in both culture conditions (Fig. 1f, Supplementary Fig. 2a). These results indicate that NMR-NS/PCs can proliferate while remaining in an undifferentiated state using the neurosphere or adherent culture protocol.

NMR-NS/PCs proliferate slower than mouse-NS/PCs
Proliferation of NMR-NS/PCs in adherent culture was compared with that of mouse-NS/PCs under the same temperature and oxygen conditions (32°C, 3% O 2 ). NMR-NS/PCs proliferated slower than mouse-NS/PCs (Fig. 2a). The doubling time of NMR-NS/PCs was 120 h, which was 1.5 times longer than that of mouse-NS/PCs (80 h) (Fig. 2b). Cell cycle analysis by flow cytometry showed that a higher percentage of NMR-NS/PCs than mouse-NS/PCs was in G0/G1 phase, suggesting that NMR-NS/PCs have a longer G1 phase or more NMR-NS/PCs are in G0 phase (Fig. 2c, d). NMR-NS/PCs could not proliferate at 37°C and in 21% O 2 , the standard culture condition for mouse-NS/PCs (Supplementary Fig.  2b). These results indicate that NMR-NS/PCs have a lower proliferation potential than mouse-NS/PCs.

NMR-NS/PCs can differentiate into three neural lineages
To induce differentiation, NMR-NS/PCs grown in neurosphere or adherent culture conditions were plated onto poly-L-ornithine/fibronectin-coated plates and cultured for 10 days without bFGF and EGF as previously described in mice (Fig. 3a) [33]. Immunocytochemistry showed that differentiated cells were positive for neuron markers (TUBB3 and MAP2), astrocyte markers (GFAP and S100B), and oligodendrocyte markers (O4 and OSP) (Fig. 3b, Supplementary Fig. 2c). These data suggest the possibility that cultured NMR-NS/PCs can differentiate into three neural lineages.

NMR-NS/PCs are resistant to DNA damage induced by γirradiation
To evaluate the response of NMR-NS/PCs to DNA damage, we performed γ-irradiation ( Supplementary Fig. 3a). Immunocytochemistry and quantification of γH2AX, a DSB marker [34], showed that the signal intensity of γH2AX at 1 h after γ-irradiation was significantly lower in NMR-NS/PCs than in mouse-NS/PCs, suggesting that DNA damage is less severe in NMR-NS/PCs than in mouse-NS/PCs (Fig. 4a, b). We then examined changes in the foci numbers of 53BP1 and pATM, which are DNA damage response markers, in nuclei after γirradiation [35,36]. Notably, more 53BP1 foci had formed in NMR-NS/PCs than in mouse-NS/PCs at 10 min after γ-irradiation, and the number of 53BP1 foci remained high in NMR-NS/PCs until 24 h (Fig. 4a, c). γH2AX and 53BP1 tended to be colocalized more frequently in NMR-NS/PCs than in mouse-NS/PCs at 10 min, 30 min, and 1 h after γ-irradiation (Fig. 4a). Similarly, more pATM foci had formed in NMR-NS/PCs than in mouse-NS/PCs at 30 min and 1 h after γ-irradiation (Fig. 4d, Supplementary Fig. 3b). These results suggest that DNA repair is initiated faster in NMR-NS/PCs than in mouse-NS/PCs. After 72 h, γH2AX signals decreased in both NMR-and mouse-NS/PCs, and 53BP1 and pATM foci had returned to the levels observed before γ-irradiation ( Fig. 4b-d). Consistent with the data suggesting a fast DNA damage response, NMR-NS/PCs were less prone to dying upon γ-irradiation than mouse-NS/PCs. At 24 h after exposure to 10 Gy of γradiation, the percentage of dead NMR-NS/PCs was not significantly increased (Fig. 4e). At 72 h after exposure  to 10 Gy of γ-radiation, about 20% of NMR-NS/PCs were dead, in contrast with about 50% of mouse-NS/PCs (Fig. 4e). p53 responds to DNA damage and induces expression of genes involved in both cell cycle arrest and apoptosis [37,38]. At 24 h after γ-radiation, RT-qPCR analysis of genes downstream of p53 was performed. Among cell death-related genes, expression of BCL2associated X, apoptosis regulator (BAX) was upregulated in both NMR-and mouse-NS/PCs upon γ-irradiation, whereas expression of phorbol-12-myristate-13-acetate- Fig. 4 Comparison of the DNA damage response after γ-irradiation between neural stem/progenitor cells from naked mole-rats (NMR-NS/PCs) and mice (mouse-NS/PCs). a Immunofluorescence images of phosphorylated histone H2AX (γH2AX; magenta) and tumor protein p53-binding protein 1 (53BP1; green) in NMR-and mouse-NS/PCs exposed to 10 Gy of γ-radiation or left untreated. Blue, Hoechst 33342. White arrowheads indicate positive cells with >10 53BP1 foci per nuclei. Scale bars: 10 μm. b-d Quantification of the average signal intensity of γH2AX per nuclei (b), cells with >10 53BP1 foci (c), and cells with >10 phosphorylated ATM serine/threonine (pATM) foci (d). Data are mean ± standard deviation (SD) from n = 3 biological replicates. ***P < 0.001; ****P < 0.0001; N.S., not significant, two-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparisons test. e Quantification of cell death using propidium iodide (PI)/Hoechst staining after γ-irradiation. Data are mean ± SD from n = 3 biological replicates. ***P < 0.001; N.S., not significant, one-way ANOVA followed by Dunnett's multiple comparison test. f Relative expression of genes downstream of p53 at 24 h after γ-irradiation determined by reverse transcription-quantitative PCR. Fold changes were calculated relative to the untreated control. Data are mean ± SD from n = 3 biological replicates. **P < 0.01; ***P < 0.001; ****P < 0.0001; N.S., not significant, unpaired t-test induced protein 1 (PMAIP1, also known as NOXA) and Fas cell surface death receptor (FAS) was upregulated less in NMR-NS/PCs than in mouse-NS/PCs. In terms of cell cycle arrest-related genes, cyclin-dependent kinase inhibitor 1A (CDKN1A), protein phosphatase, Mg 2+ / Mn 2+ -dependent 1D (PPM1D, also known as WIP1), and MDM2 proto-oncogene (MDM2) were upregulated in NMR-NS/PCs (Fig. 4f). These differences in expression of genes downstream of p53 are consistent with fewer NMR-NS/PCs dying upon γ-irradiation. Taken together, our results indicate that NMR-NS/PCs are more resistant to γ-irradiation than mouse-NS/PCs.

Discussion
In this study, we successfully isolated and cultured NS/ PCs from the SVZ of neonatal NMRs. Compared with mouse-NS/PCs, NMR-NS/PCs proliferated significantly slower and more cells were in G0/G1 phase. Notably, NMR-NS/PCs were more resistant to γ-irradiation than mouse-NS/PCs. In this study, we compared the characteristics of NS/ PCs derived from the SVZs of NMRs and mice at postnatal days 1-2. A previous study reported that the numbers of SOX2-positive cells in the SVZs of mouse and NMR neonates are similar [39]. On the other hand, Orr et al. showed that the brains of postnatal NMRs are more developed than those of mice and have a clearly laminated hippocampus and myelinated white matter tracts, similar to brains of neonatal primates. On the other hand, postnatal NMR brains mature slower than mouse brains. Thus, the developmental stages of NMR SVZs and the properties of NSCs derived from them must be compared with those of mice in more detail in the future, not only in the neonatal stage but also in the fetal stage.
Proliferation of NMR skin fibroblasts is reportedly suppressed by induction of early contact inhibition via INK4A activation due to signaling of high molecular mass hyaluronan [10,40]. On the other hand, we previously showed that NMR iPS cells have a lower proliferative capacity than mouse iPS cells [11]. In this study, we revealed that proliferation of NMR-NS/PCs is lower than that of mouse-NS/PCs. These two types of NMR stem cells do not exhibit an early contact inhibition phenotype, suggesting that cell cycle progression is negatively regulated in several types of NMR cells. Further studies of cell cycle control in NMR cells are required to better understand longevity and cancer resistance in this species.
Genome instability is closely related to aging and cancer. It has been suggested that long-lived mammals have a high DNA repair capacity [41]. In this study, we found that the DNA damage response of NMR-NS/PCs has unique characteristics. In the early stage, NMR-NS/PCs showed less DNA damage (signal intensity of γH2AX) and more repair sites (numbers of 53BP1 and pATM foci) than mouse-NS/PCs. Previous reports suggested that some DNA repair pathways are highly efficient in NMRs due to high poly ADP-ribosylation activity [14,16,42]. Poly ADP-ribosylation is an essential process in the early stages of the DNA damage response [43]. Therefore, high poly ADP-ribosylation activity may contribute to efficient DNA repair in NMR-NS/PCs. In the late stage, irradiated NMR-NS/PCs were resistant to death. NMR fibroblasts are less prone to undergo acute death than mouse fibroblasts upon γ-irradiation [44]. The low ability to induce death of NMR-NS/PCs upon DNA damage may contribute to the long-term maintenance of the stem cell pool during the long lifespan of NMRs.
In contrast with NMR cells, lymphocytes from cancerresistant elephants are highly vulnerable to γ-irradiation and prone to death [45]. This is thought to be one of the mechanisms underlying cancer resistance in elephants via efficiently eliminating mutant cells with damaged DNA from tissues. Hence, the low ability to induce death of NMR cells after induction of DNA damage by γ-irradiation might be a double-edged sword because it may leave mutant cells with damaged DNA in the tissues of NMRs. Therefore, NMR cells may have a very accurate DNA repair system that suppresses the appearance of mutant cells without killing damaged cells. Further analyses, such as comet assays, karyotyping, or genomic sequencing, are required to determine whether accurate DNA repair occurs in NMR cells that survive after DNA damage. It is also necessary to analyze whether poly ADP-ribosylation affects efficient DNA repair in NMR-NS/PC as well as in NMR-fibroblasts [14,16,42].

Conclusions
In this study, we successfully isolated and cultured NS/ PCs from the SVZ of neonatal NMRs. The slow proliferation and fast DNA damage response of NMR-NS/PCs may contribute to the delayed-aging phenotype in brain tissues of NMRs by preventing stem cell depletion and maintaining the stem cell pool. Future research of NMR somatic stem cells may lead to the elucidation of novel mechanisms that prevent stem cell exhaustion and dysfunction during aging.