In vivo characterization of primitive hematopoietic cells in clonal ginbuna crucian carp (Carassius auratus langsdorfii)

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Veterinary Immunology and Immunopathology 126 (2008) 74–82 www.elsevier.com/locate/vetimm

In vivo characterization of primitive hematopoietic cells in clonal ginbuna crucian carp (Carassius auratus langsdorfii) Isao Kobayashi *, Hiroko Kusakabe, Hideaki Toda, Tadaaki Moritomo, Tomoko Takahashi, Teruyuki Nakanishi Department of Veterinary Medicine, Nihon University, Kameino 1866, Fujisawa, Kanagawa, Japan Received 10 March 2008; received in revised form 12 June 2008; accepted 23 June 2008

Abstract Primitive hematopoietic cells in mammalian bone marrow are purified by flow cytometry using Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), because these dyes efflux activities of hematopoietic cells widely conserved in mammals. Hematopoietic stem cells (HSCs) are identified as side population (SP) cells, characterized by specific Hoechst efflux pattern in flow cytometric analysis. We previously demonstrated that SP cells from teleost body kidney (BK) had the HSC activity by a transplantation experiment using clonal ginbuna crucian carp (Carassius auratus langsdorfii). In the present study, to isolate HSCs and hematopoietic progenitor cells (HPCs) from teleosts using Hoechst and Rho, we compared the hematopoietic activity of Rhonegative (Rho ) cells with that of SP cells by ginbuna transplantation experiments. Rho cells were clearly identified from ginbuna BK, and the majority of these cells (85%) showed a non-SP phenotype. Transplantation experiments showed that long-term repopulating activity (HSC activity) of Rho cells was lower than that of SP cells, while Rho cells had higher short-term repopulating activity (HPC activity) than SP cells. These results suggest that Rho cells in ginbuna BK contain various stages of hematopoietic cells, while SP cells are highly enriched for HSCs, and that these dyes are useful for purification of HSCs and HPCs in teleosts. # 2008 Elsevier B.V. All rights reserved. Keywords: Hematopoietic stem cells; Hematopoietic progenitor cells; Rhodamine-123; Hoechst 33342; Ginbuna; Transplantation

1. Introduction Hematopoietic stem cells (HSCs) produce hematopoietic progenitor cells (HPCs) for all hematopoietic lineages and are the source of the cellular elements of the blood over the life span of the organism. In murine study, HSCs and HPCs are functionally characterized by their ability to repopulate in recipient animals (Zhong et al., 1996). HSCs are generally in a quiescent

* Corresponding author. Tel.: +81 466 84 3443; fax: +81 466 84 3380. E-mail address: [email protected] (I. Kobayashi). 0165-2427/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2008.06.008

state of the cell cycle and slowly proliferate. In contrast, HPCs actively proliferate and differentiate, whereas their potential of proliferation is more limited than HSCs. Therefore, HSCs reveal delayed but long-term hematopoietic reconstitution in lethally irradiated hosts, while transplantation of HPCs results in early but unsustained reconstitution (Zhong et al., 1996; Osawa et al., 1996; Mayani et al., 2003). Thus, transplantation study to test the repopulating activity of donor cells has become the gold standard for the characterization of HSCs and HPCs. Hematopoiesis in teleosts is very similar to that in mammals. Definitive blood cell lineages in teleosts show a high degree of similarity at the morphological

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and molecular level to their mammalian counterparts (de Jong and Zon, 2005). Zebrafish (Danio rerio) has recently emerged as a powerful genetic model of hematopoiesis (de Jong and Zon, 2005; Bahary and Zon, 1998). Although several transplantation model systems of zebrafish have been established (Traver et al., 2003, 2004; Langenau et al., 2004), in vivo functional analysis of hematopoietic cells in zebrafish is difficult because of their small body size and the lack of inbred strains. We developed a transplantation model system for detecting HSCs using clonal ginbuna crucian carp (Carassius auratus langsdorfii, S3n strain) and ginbuna-goldfish (Carassius auratus) hybrids (S4n strain) (Nakanishi and Ototake, 1999; Moritomo et al., 2004). Ginbuna is a triploid fish and principally reproduces gynogenetically. The S4n hybrid possesses four sets of chromosomes, three from S3n and one from goldfish, and is genetically tolerant of S3n cells (Fischer et al., 1999). When mononuclear cells (5  106 cells) from an S3n kidney, the main hematopoietic organ in teleosts, were injected into S4n recipients, all types of donor-derived blood cells were sustained in the recipient blood for over 1 year (Kobayashi et al., 2006). This observation clearly demonstrated the presence of HSCs in teleost kidney, and we can evaluate the HSC activity of donor cells by this ‘‘long-term repopulation assay’’. Furthermore, we recently developed a transplantation model system for detecting HPCs using lethally irradiated ginbuna. Injection of kidney hematopoietic cells (5  106 cells) into recipient ginbuna (OB1 strain) that were exposed to a lethal dose of X-rays (25 Gy) resulted in the reconstitution of the recipient hematopoietic system within 9 days after injection and the rescue of recipient fish (Kobayashi et al., 2008a). This early hematopoietic reconstitution in lethally irradiated ginbuna is derived from HPCs, and therefore we can also evaluate the HPC activity of donor cells by this ‘‘short-term repopulation assay’’. Thus, these transplantation model systems using clonal ginbuna are useful for the identification of HSCs and HPCs in teleosts. Purification of primitive hematopoietic cells is essential for the molecular biological analysis of differentiation capabilities of hematopoietic cells. The availability of methods to isolate HSCs and HPCs in teleosts is very limited, and therefore the molecular mechanisms of hematopoietic differentiation in teleosts are still poorly understood. Enrichment techniques for mammalian HSCs include the use of flow cytometry (FCM) with Hoechst 33342 (Hoechst) and rhodamine123 (Rho). HSCs are identified on the basis of their high dye efflux activity (Wolf et al., 1993; Leemhuis et al., 1996). Hoechst efflux activity of HSCs is attributed to

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their expression of an ATP binding cassette (ABC) transporter, ABCG2/Abcg2 (Zhou et al., 2001, 2002), and these Hoechst-low HSCs are identified as side population (SP) cells (Goodell et al., 1996, 1997). In contrast, Rho efflux activity of HSCs is attributed to another ABC transporter, P-glycoprotein (P-gp) (Uchida et al., 2002), and HSCs are also present in the Rhonegative (Rho ) population (Spangrude and Johnson, 1990; Orlic et al., 1993). Since the dye efflux activities of HSCs are widely conserved in mammals, Hoechst and Rho can be used for the purification of HSCs from a variety of mammals (Goodell et al., 1997; Baum et al., 1992; Mahmud et al., 2001). Our previous study showed that SP cells were present in ginbuna body kidney (BK) and had long-term repopulating activity in S4n recipients (Kobayashi et al., 2007). Furthermore, our recent study showed that Hoechst efflux activity of zebrafish (Danio rerio) kidney SP cells was attributed to the expression of a homologous protein of human ABCG2, zAbcg2a (Kobayashi et al., 2008b). These observations indicate that Hoechst efflux activity of HSCs is conserved in teleosts. On the other hand, there is no information about Rho efflux activity of teleost HSCs or about the hematopoietic activity of Rho cells in teleost kidney. In murine bone marrow, the HSC frequency of Rho cells was lower than that of SP cells (Uchida et al., 2004). In addition, P-gp mRNA is strongly expressed in myeloid and lymphoid progenitors as well as multipotent stem/progenitor cells (Drach et al., 1992; Chaudhary and Roninson, 1991; Sorrentino et al., 1995), indicating that Rho cells contain various stages of hematopoietic cells. On the other hand, SP cells are highly enriched for HSCs (Goodell et al., 1997; Zhou et al., 2001). In the present study, to isolate HSCs and HPCs from teleost kidney using Hoechst and Rho, we examined the functional properties of SP cells and Rho cells in ginbuna BK. We first isolated Rho cells from ginbuna BK, and examined the relationship between Rho cells and SP cells by FCM analysis. We also examined the expression of lymphoid-lineage (LymLin) surface markers, such as CD4, CD8a, and IgM, on these cells using monoclonal antibodies (mAb). In addition, we compared the hematopoietic activity of Rho cells with that of SP cells by long- and short-term repopulation assays. 2. Materials and methods 2.1. Fish maintenance Triploid clonal ginbuna (S3n and OB1 strain, 15– 30 g, 1–2 years of age) and tetraploid hybrids (S4n

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strain, 50–60 g, 2–4 years of age) were used. The S3n and OB1 strain have been maintained in the laboratory for more than 10 years with repeat bleeding. S4n hybrids were produced by insemination of triploid S3n clone eggs with goldfish sperms as previously described (Nakanishi, 1987). Fish were kept at 25  1 8C in a recirculation system with filtered water and were fed pelleted dry food once a day. 2.2. Cell preparation Cells were obtained from ginbuna body (mesonephric, trunk, posterior) kidney and head (pronephric, anterior) kidney (HK) as previously described (Kobayashi et al., 2006). Briefly, ginbuna were anesthetized with 0.01% benzocaine (Sigma, St. Louis, MO, USA) and were killed by decapitation before tissue collection. BK and HK were dissected, and cells were respectively obtained by macerating the tissues in 5 ml of ice-cold Hank’s balanced salt solution (HBSS) containing with 2% of fetal bovine serum (FBS). Cells were pelleted by centrifugation (400  g for 5 min). After discarding the supernatant, the pellet was gently mixed with 1 ml of distilled water by pipetting to lyse the erythrocytes by osmotic shock. Subsequently, 1 ml of 2 HBSS was added, and cells were washed with HBSS by centrifugation. 2.3. Flow cytometry After resuspended at a density of 106 cells/ml in HBSS, cells were stained with rhodamine-123 (Rho, Sigma) according to the procedure for murine bone marrow cells with modifications (McAlister et al., 1990). After preliminary staining with 0.05, 0.1, 0.2, and 0.3 mg/ml Rho for 20, 30, and 40 min at 25 8C, a staining procedure with 0.1 mg/ml Rho for 20 min was used in the majority of experiments. For inhibition experiments, cells were stained with Rho in the presence of 5 mM reserpine (Sigma). For doublestaining analysis, cells were stained with 7.5 mg/ml Hoechst 33342 (Hoechst, Molecular Probe, Eugene, OR, USA) for 70 min at 25 8C. After then, Rho was added, and cells were incubated for another 20 min. For antibodies staining, non-staining BK cells were resuspended at a density of 107 cells/ml in HBSS. The cocktail of Lym-Lin mAb, rat anti-ginbuna CD4, rat anti-ginbuna CD8a, and mouse anti-ginbuna IgM (mouse ascites), was added at 1:104 dilutions (0.1– 1 mg/107 cells), and cells were incubated for 45 min on ice. After being washed three times with HBSS, cells were resuspended and incubated with a 1:200 dilution

(approximately 10 mg/107 cells) of biotin-conjugated goat anti-rat IgG and anti-mouse IgG/M antibodies (Jackson Immunoresearch, Labs., Inc., West Grove, PA) for 30 min on ice and then re-washed further twice. Cells were stained with a 1:20 dilution of streptavidinPE conjugated (Serotec, Oxford, UK) for 20 min on ice then re-washed further twice. After being washed, cells were resuspended at a density of 106 cells/ml in HBSS and stained with both Hoechst and Rho. After staining, cells were washed by centrifugation, adjusted to 107 cells/ml in HBSS, and kept on ice until use. Just before FCM analysis, propidium iodide (PI, Molecular Probe) solution was added at a final concentration of 2 mg/ml to identify non-viable cells. FCM analysis and cell sorting were performed by EPICS ALTRA (Beckman Coulter, Fullerton, CA, USA) as previously described (Kobayashi et al., 2007). 2.4. Long-term repopulation assay After being sorted from S3n ginbuna BK, cells (3  104) were resuspended in 0.1 ml of HBSS and were injected into S4n recipients via caudal vessels. In an attempt to stimulate donor cell proliferation, S4n recipients were severely bled to induce anemia as previously described (Kobayashi et al., 2006). For ploidy analysis, peripheral blood leukocytes from the recipients were stained with Hoechst (10 mg/ml) and analyzed by EPICS ALTRA as previously described (Kobayashi et al., 2006). 2.5. Short-term repopulation assay X-ray irradiation was performed as previously described (Kobayashi et al., 2008a). Briefly, two ginbuna (OB1 strain) were simultaneously anesthetized, placed into a humidified chamber containing anesthetizing water, and irradiated with 25 Gy of X-rays (high stability constant potential X-ray systems, Philips Medical Systems, Best, The Netherlands) at a dose rate of 2.5 Gy/min. Because our previous study showed that almost all leukocytes were depleted from BK at 5 days after 25 Gy irradiation (Kobayashi et al., 2008a), cells (1.5  104) obtained from non-irradiated OB1 ginbuna were injected into irradiated ginbuna at 5 days after irradiation. As a control, 0.1 ml of HBSS was injected into irradiated ginbuna. After injection, these fishes were kept in aquarium water containing 0.3–0.5% NaCl for preventing infection. At 15 days after injection (20 days after irradiation), BK cells were obtained from the recipient fish as described in Section 2.2. FCM analyses and cell counts of BK cells were performed by

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FACS Canto (Becton Dickinson, Hialeah, FL, USA) as previously described (Kobayashi et al., 2008a). 3. Results 3.1. Isolation of Rho cells from ginbuna body kidney To isolate Rho cells from ginbuna BK, BK cells were stained with Rho and were analyzed by FCM. When BK cells stained with Rho were displayed in a rhodamine-123 (green fluorescence) vs. SS (side scatter) dot-plot, high green fluorescence intensity was observed in the majority of BK cells (Fig. 1A) compared to non-staining BK cells (Fig. 1B). In this staining profile, the Rho population was identified as a small streak of cells with very low SS (gated region in Fig. 1A). The percentage of Rho cells in BK cells was 0.64%  0.08 (n = 4). Rho cells were sorted and morphologically examined (Fig. 1C). Rho cells were predominantly composed of two types: cells having a thin-layered cytoplasm and a round nucleus like an SP cell (Kobayashi et al., 2007) (Fig. 1C, a), and cells

Fig. 1. Flow cytometric analyses of ginbuna body kidney (BK) cells. (A) BK cells stained with rhodamine-123 (Rho) are displayed in a rhodamine-123 vs. SS dot-plot. Gated region shows the Rho population. (B) Non-staining BK cells are shown in a rhodamine-123 vs. SS dot-plot. (C) Morphological analysis of Rho cells is shown. Rho cells were smeared and stained with May–Gruenwald Giemsa solution. There are two types of cells in the Rho population (‘a’ and ‘b’). Scale bar indicates 5 mm. (D) BK cells stained with Rho in the presence of 5 mM reserpine are shown in a rhodamine-123 vs. SS dot-plot. Gated region shows the Rho population.

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having relatively abundant cytoplasm like a blast cell (Fig. 1C, b). To investigate whether low green fluorescence of Rho cells is mediated by ABC transporters, BK cells were stained with Rho in the presence of reserpine, an inhibitor of ABC transporters (Bhat et al., 1995). As shown in Fig. 1D, the percentage of Rho cells was significantly decreased by reserpine treatment (0.33%  0.08, n = 4, p < 0.05), suggesting that the low green fluorescence of Rho cells reflects the activity of ABC transporters. 3.2. Double-staining analysis with Hoechst and Rho To examine the relationship between SP cells and Rho cells, BK cells were stained with both Hoechst and Rho, and were analyzed by FCM. Typical result of double-staining analysis was shown in Fig. 2. SP cells are characterized by specific fluorescent pattern in a Hoechst Red vs. Hoechst Blue dot-plot (Goodell et al., 1997; Kobayashi et al., 2007). When Rho cells were displayed in a Hoechst Red vs. Hoechst Blue dot-plot, two distinct populations (SP and non-SP) were observed (Fig. 2A and B). Although the majority of Rho cells

Fig. 2. Double-staining analysis of body kidney (BK) cells. BK cells were stained with both Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), and were analyzed by flow cytometry. (A) BK cells were displayed in a rhodamine-123 vs. SS dot-plot. (B) After gating the Rho population, Hoechst Red vs. Hoechst Blue dot-plot was then obtained for the gated region. Rho cells are subdivided into SP and non-SP populations. (C) BK cells were displayed in a Hoechst Red vs. Hoechst Blue dot-plot. (D) After gating the SP population, rhodamine123 vs. SS dot-plot was then obtained for the gated region. SP cells are subdivided into Rho and Rho+ populations.

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showed a non-SP phenotype, 15.0% of Rho cells fell into the SP population. In contrast, when SP cells were displayed in a rhodamine-123 vs. SS dot-plot, 27.1% of SP cells showed a Rho phenotype (Fig. 2C and D). The percentage of SP Rho cells in BK cells was 0.097%  0.034 (n = 4). We also performed double-staining analysis in head kidney (HK) cells, which hardly contain SP cells (Kobayashi et al., 2007). As shown in Fig. 3A, the percentage of Rho cells in HK (1.21%  0.27, n = 4) was significantly higher than that in BK ( p < 0.05). However, when Rho cells from HK were displayed in a Hoechst Red vs. Hoechst Blue dot-plot, none of the Rho cells showed an SP phenotype (Fig. 3B). 3.3. Phenotypic characterization of SP cells and Rho cells Because morphological analysis showed that both SP cells and Rho cells contain lymphocyte-like cells, we next examined the expression of Lym-Lin antigens (CD4, CD8a, and IgM) on these cells. Fig. 4 shows typical results of fluorescence profiles of BK cells stained with a cocktail of Lym-Lin antibodies, Hoechst, and Rho. Since SP cells were resolved by only Hoechst Red fluorescence (Fig. 2C), we defined the Hoechst Red-low population as an SP population in this analysis (Fig. 4A). The percentages of the SP LymLin+ and SP Lym-Lin populations were 0.01% and 0.25%, respectively (Fig. 4A), indicating that only 3.8% of SP cells expressed Lym-Lin antigens. In contrast, the percentages of the Rho Lym-Lin+ and Rho Lym-Lin populations were 0.23% and 0.54%, respectively (Fig. 4B), indicating that 29.9% of Rho cells expressed Lym-Lin antigens. These results indicate that SP cells contain only small numbers of

Fig. 4. Expression of lymphoid-lineage (Lym-Lin) antigens on SP cells and Rho cells. Body kidney (BK) cells were stained with the cocktail of Lym-Lin (CD4, CD8a, and IgM) antibodies, Hoechst 33342, and rhoddamine-123, and were analyzed by flow cytometry. Typical dot-plot profiles of Hoechst Red vs. Lym-Lin (A) and rhodamine-123 vs. Lym-Lin (B) are shown. Similar results were obtained from three independent experiments.

mature lymphocytes, although large numbers of lymphocytes were present in the Rho population. 3.4. Long-term repopulation assay for SP cells and Rho cells To compare long-term repopulating activity (HSC activity) of SP cells and Rho cells, the same number of cells (30,000 cells) obtained from S3n BK were injected into S4n recipients. Nine months after injection, ploidy analysis was performed in these recipients. The lifespan of granulocytes in ginbuna are only 5 days (Fischer et al., 1998), and therefore donor-derived granulocytes in recipient peripheral blood are the best indicator of the repopulating activity of donor cells. As shown in Table 1, the percentage of donor-derived granulocytes in R1 (transplanted with SP cells) was 4.6%. Similar or higher percentages were observed in our previous study (Supplemental Table 1). In contrast, the percentages of donor-derived granulocytes in R2-5 (transplanted with Rho cells) were lower than that in R1 (0.9–2.5%).

Table 1 Long-term repopulation assay for SP cells and Rho cells

Fig. 3. Double-staining analysis of head kidney (HK) cells. HK cells were stained with both Hoechst 33342 (Hoechst) and rhodamine-123 (Rho), and were analyzed by flow cytometry. (A) HK cells were displayed in a rhodamine-123 vs. SS dot-plot. (B) After gating the Rho population, Hoechst Red vs. Hoechst Blue dot-plot was then obtained for the gated region. All of Rho cells in HK show a non-SP phenotype.

Donor type

Recipient

Transplanted cells (#)

Donor-derived granulocytes (9 months) (%)

SP Rho Rho Rho Rho

R1 R2 R3 R4 R5

30,000 30,000 30,000 30,000 30,000

4.6 0.9 1.5 1.1 2.5

After being sorted from S3n body kidney, same number (30,000) of SP cells and Rho cells were injected into S4n recipients (R1-5). Nine months after injection, percentages of donor-derived granulocytes were examined in these recipients by ploidy analysis.

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These results suggest that the HSC activity of Rho cells is lower than that of SP cells. 3.5. Short-term repopulation assay for SP cells and Rho cells We also compared short-term repopulating activity (HPC activity) of SP cells and Rho cells. The same number of SP cells and Rho cells (15,000 cells) obtained from OB1 BK were injected into lethally irradiated syngenic recipients. 15 days after injection (20 days after irradiation), the survival rate of these recipient fishes (SP fish and Rho fish) and nontransplanted fish (control fish) were examined. As shown in Table 2, the survival rate of Rho fish (75%, n = 4) was higher than that of SP fish (37.5%, n = 8) and control fish (14.3%, n = 7). Our previous study showed that hematopoiesis did not recover without transplantation in lethally irradiated ginbuna, and that the renewal potential of donor hematopoietic cells could be evaluated by the number of BK cells in recipient fish (Kobayashi et al., 2008a). At 15 days after injection, BK cells were obtained from SP fish and Rho fish, and were analyzed by FCM. In normal ginbuna BK, three distinct populations, lymphoid (L; FSlow, SSlow), precursor (P; FShigh, SSlow/mid), and granulocyte (G; FSmid, SShigh), were identified by the scatter profile (Fig. 5A, right lane). In SP fish and Rho fish, recoveries of lymphoid and precursor population were observed (Fig. 5A, left and middle lane). However, the total number of BK cells in Rho fish was higher than that of SP fish (Fig. 5B). These results suggest that the HPC activity of Rho cells is higher than that of SP cells. 4. Discussion In the present study, we identified Rho cells from ginbuna kidneys. Transplantation experiments showed that the long-term repopulating activity of Rho cells Table 2 Survival rates of lethally irradiated recipient ginbuna Recipient

No. of survival /total

Survival (%)

SP fish Rho fish Control fish

3/8 3/4 1/7

37.5 75 14.3

Same number (15,000) of SP cells and Rho cells were injected into lethally irradiated ginbuna at 5 days after irradiation. Survival rates of these recipient fish (SP fish and Rho fish) and non-transplanted fish (control fish) were examined at 15 days after injection (20 days after irradiation)

Fig. 5. Short-term repopulation assay for SP cells and Rho cells. After injection of SP cells and Rho cells into lethally irradiated ginbuna, body kidney (BK) cells were obtained from the recipient fish (SP fish and Rho fish) at 15 days after injection. (A) Scatter profiles of BK cells from the SP fish (left lane), Rho fish (middle lane), and normal fish (right lane) are shown. Gated regions show a lymphoid (L; FSlow, SSlow), precursor (P; FShigh, SSlow/mid), and granulocyte (G; FSmid, SShigh) population, respectively. (B) Numbers of BK cells from SP fish (left lane) and Rho fish (right lane) are shown. The values are the means of three fish  S.D.

was lower than that of SP cells, whereas Rho cells had higher short-term repopulating activity than SP cells. These results suggest that Rho cells in ginbuna BK, like those in mammalian bone marrow, contain various stages of hematopoietic cells including HSCs and HPCs, whereas SP cells are highly enriched for HSCs. Both P-gp and Abcg2 have been associated with resistance to chemotherapeutic drugs in cancer cells (Pastan and Gottesman, 1991; Doyle et al., 1998), whereas the normal physiologic roles of these transporters are unclear. In mice, enforced expression of the P-gp gene in bone marrow cells resulted in marked amplification of repopulating cells, suggesting that Pgp plays a role in the expansion of hematopoietic cells (Bunting et al., 1998, 2000). On the other hand, overexpression of human ABCG2 in murine bone marrow cells significantly blocked hematopoietic development, leading to speculation that Abcg2 plays a role in early stem cell self-renewal by blocking differentiation (Zhou et al., 2001). Although the functional roles of such transporters in hematopoietic cells are still unclear, the conservations of Hoechst and Rho efflux activities in hematopoietic cells among vertebrates may reflect an important role in hematopoiesis.

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In this study, we found some differences between SP cells and Rho cells besides their effects on hematopoietic activity. First, the percentage of Rho cells in BK was approximately 2-times higher than that of SP cells (Fig. 2). Ginbuna SP cells are a more homogeneous population than Rho cells with respect to morphology and cell-surface phenotype. The high percentage of Rho cells in BK probably reflects the functional heterogeneity of this population. Second, SP cells and Rho cells showed different sensitivities to inhibitors of ABC transporters (reserpine and verapamil). Our previous study showed that ginbuna SP cells were eliminated by verapamil treatment (Kobayashi et al., 2007), but not by reserpine treatment (data not shown). In contrast, Rho cells were sensitive to reserpine (Fig. 1D), but not to verapamil (data not shown). These observations suggest that different transporters are involved in Hoechst and Rho efflux activities in teleosts, as is the case in mammals (Zhou et al., 2001). However, it is unclear why Rho efflux activity in ginbuna was not blocked by verapamil, which is a calcium channel blocker and known to be an inhibitor of P-gp. Hoechst efflux activity of ginbuna SP cells was blocked by 500 mM of verapamil (Kobayashi et al., 2007), which is 10-fold higher concentration than the concentration used in the murine procedure (Goodell et al., 1996), suggesting that hematopoietic cells are more resistant to verapamil in teleosts than in mammals. Third, number of Rho cells is greater in HK than BK (Fig. 3), while SP cells are found only in BK (Kobayashi et al., 2007). Although HK and BK are the main hematopoietic organs in teleosts (Kobayashi et al., 2006, 2008a), BK has larger number of renal tubules and glomeruli than HK (Press and Evensen, 1999; Fange, 1986). We previously showed that SP cells in ginbuna and zebrafish were localized on the surface of renal tubules in BK and tightly adherent to renal tubule epithelial cells (Kobayashi et al., 2008b). This suggests that renal tubules in teleosts are a key component of the stem cell niche, the microenvironment in which stem cells are maintained. These observations suggest that BK is the major source of HSCs in teleosts. Although the mechanisms of the maintenance of HSCs and HPCs in teleost kidneys are unclear, the different percentages of Rho cells in HK and BK also suggest that there are different microenvironments surrounding HPCs as well as HSCs in HK and BK. In mammals, it is known that primitive HSCs are highly enriched in the SP Rho population (Uchida et al., 2004), and in some cases, both Hoechst and Rho are used for purification of HSCs (Wolf et al., 1993; Bertoncello and Williams, 2004). Although our

transplantation model systems using clonal ginbuna proved to be useful for measuring HSC and HPC activity of donor cells, large number of donor cells are necessary for transplantation experiments. In the present study, we could not obtain a sufficient number of SP Rho cells from ginbuna BK because of their low percentage, and therefore the hematopoietic activity of SP Rho cells could not be examined. However, the high conservations of Hoechst and Rho efflux activities in teleost hematopoietic cells strongly suggest that teleost HSCs also show an SP Rho phenotype. If so, the double-staining method using both Hoechst and Rho may be useful for further purification of HSCs in ginbuna. In addition, the high HPC activity of Rho cells suggests that a part of the Rho population, LymLin non-SP Rho , contains many HPCs. Although further functional analyses are necessary to determine the HSC and HPC frequencies in these populations, Hoechst and Rho, combined with Lym-Lin mAb, may be useful for purification of HSCs and HPCs from ginbuna. The properties of HSCs, which are altered when they begin to differentiate, are of great interest because the molecules involved may serve as indicators of mechanisms that determine their stem cell activity. A major obstacle in the analysis of HSCs is separating HSCs from HPCs and mature blood cells. Purification of HSCs in mammals has been traditionally achieved by the establishment of mAb to recognize HSCs (Wognum et al., 2003). CD34 is commonly accepted as a marker for HSCs in mammals (Berenson et al., 1988; Spangrude and Johnson, 1990; Bruno et al., 1999; Verfaillie et al., 1990). However, murine HSCs have been recognized as CD34-negative (Osawa et al., 1996; Goodell et al., 1997). Instead of CD34, stem cell antigen-1 (Sca-1) and c-kit are generally used for identification of murine HSCs (Osawa et al., 1996; Spangrude et al., 1988). Thus, the expressions of surface markers in HSCs differ between species. On the other hand, previous studies (Baum et al., 1992; Goodell et al., 1997; Kobayashi et al., 2007) and the present results provide evidence that the dye efflux activities of hematopoietic cells are highly conserved in vertebrates. This finding suggests that Hoechst and Rho are useful for purification of hematopoietic cells from other vertebrate species (e.g. Xenopus tropicalis). In conclusion, Hoechst and Rho can be used for the isolation of not only HSCs but also HPCs from various species of vertebrates. This method may allow for comparative analysis of hematopoietic cells among vertebrates, and provide deeper insights into the mechanisms of stem cell self-renewal and differentiation.

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