Stem Cells, Vol. 15, No. 1, 56-62,
January 1997
© 1997 AlphaMed Press
Distinct Qualitative Differences between Normal and Abnormal Hemopoietic Stem Cells In Vivo and In Vitro
Masayo Kawamuraa,b,
Hiroko Hishaa,
Yongan Lia,
Shirou Fukuharab,
Susumu Ikeharaa
a First Department of Pathology,
b First Department of Internal Medicine, Kansai Medical University, Moriguchi-City, Osaka 570, Japan
Key Words. Hemopoietic stem cells • W/BF1 mouse • Bone marrow transplantation
Dr. Susumu Ikehara, 1st Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570, Japan.
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Abstract
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The transplantation of partially purified hemopoietic stem cells (HSCs) plus the engraftment of bone from autoimmune-prone mice ((NZW x BXSB)F1 (W/BF1) mice) induces autoimmune diseases in major histocompatibility complex (MHC)-incompatible normal C3H/HeN mice. In contrast, W/BF1 mice die of infection or anemia within three weeks due to a failure in hemopoietic reconstitution when the mice receive partially purified HSCs plus bones from normal C3H/HeN mice, although they survive more than a year without showing any symptoms of autoimmune diseases when they receive T cell-depleted bone marrow cells (without bone grafts) from normal mice. This finding suggests that abnormal HSCs can proliferate even in MHC-incompatible microenvironments, while normal HSCs cannot. This is confirmed by spleen colony-forming assays (CFU-S) on day 12, using pluripotent HSCs (P-HSCs). The P-HSCs of old (> 4 mo) W/BF1 mice (after the development of autoimmune diseases) form high CFU-S counts on day 12 even in the allogeneic C3H environment, although the P-HSCs of normal mice form high CFU-S counts only in the MHC-compatible environments. In addition, abnormal P-HSCs of autoimmune-prone mice can proliferate in vitro in collaboration with MHC-incompatible stromal cells, although normal HSCs do so in collaboration with MHC-compatible stromal cells, but not MHC-incompatible stromal cells. These findings indicate that abnormal P-HSCs are more "resilient" than normal P-HSCs.
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Introduction
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The availability of various animal models for autoimmune diseases has helped elucidate the etiopathogenesis of these diseases. We have previously found that allogeneic bone marrow transplantation (BMT) can be used to prevent insulin-dependent diabetes mellitus (IDDM), an organ-specific autoimmune disease, in nonobese diabetic (NOD) mice [1], and that the transplantation of both pancreas and BM from normal mice has a curative effect on overt diabetes in NOD mice [2]. We have also found that BMT can be used to prevent and treat systemic autoimmune diseases in BXSB [3], (NZB x NZW)F1 [4] and (NZW x BXSB)F1 [5] (W/BF1) mice; in MRL/lpr mice with abnormal radioresistant hemopoietic stem cells (HSCs), we have recently found that BMT with bone grafts (to recruit donor-derived stromal cells) prevents a relapse in autoimmune diseases [6, 7], although conventional BMT fails to prevent a relapse in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) [4]. Thus, we have been able to prevent and treat both systemic and organ-specific autoimmune diseases by conventional BMT or BMT plus bone grafts.
We have also demonstrated that the transplantation of an HSC-enriched population from autoimmune-prone mice (W/BF1 or NOD mice) into normal mice induces autoimmune diseases (SLE with idiopathic thrombocytopenic purpura (ITP) or IDDM, respectively) [8, 9]. Based on these findings, we have proposed that autoimmune diseases are stem cell disorders.
In the present study, we show that there are distinct qualitative differences between normal and abnormal HSCs both in vivo and in vitro, using the W/BF1 mouse as an autoimmune-prone mouse showing the early onset of autoimmnue diseases such as SLE and ITP [5]. Since male W/BF1 mice begin to show a high degree of proteinuria (>++) from the age of 2 mo, we compared young (<2 mo) W/BF1 mice (before the development of autoimmune diseases) with old (>4 mo) W/BF1 mice (after the development of autoimmune diseases). As female W/BF1 mice that lack the Yaa chromosome show a late onset (>10 mo) of autoimmune diseases, these mice were used as recipients in the colony-forming unit-spleen (CFU-S) assays.
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Materials and Methods
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Mice
(NZW x BXSB)F1(W/BF1)(H-2z/H-2b) mice were obtained from Kiwa Laboratory Animals (Wakayama, Japan). C3H/HeN (H-2k) mice and C57BL/6(B6)(H-2b) mice were obtained from CLEA Japan (Osaka, Japan) or Shimizu (Japan SLC; Shizuoka, Japan). All mice were maintained under specific pathogen-free conditions in our animal facility.
Purification of HSCs
BM cells were pretreated with anti-Thy1.2 monoclonal antibody (F7D5, Olac; Bicester, England) plus rabbit complement to deplete T cells (T cell-depleted bone marrow cells: TCD-BMCs). To obtain HSCs, TCD-BMCs in RPMI 1640 medium containing 10% fetal calf serum (FCS) were passed through a Sephadex G-10 column (Pharmacia Fine Chemicals; Uppsala, Sweden) to remove dead cells, macrophages and adherent cells, including stromal cells. The cells were fractionated by equilibrium density centrifugation on discontinuous Percoll (Pharmacia) gradients to remove the granulocytes and lymphocytes. For density separation, Percoll solutions were prepared in densities of 1.090, 1.073 and 1.060 g/ml. After centrifugation at 1,500 g for 30 min, the cells were collected in the following fractions: Fr. I (
< 1.063 g/ml), Fr. II (1.063 
< 1.073 g/ml), Fr. III (1.073 < 1.090 g/ml) and Fr. IV ( 
1.090 g/ml). HSCs were enriched in Fr. II, as previously described [8]. The Fr. II cells were then stained using fluorescein isothiocyanate-wheat germ agglutinin (FITC-WGA) (Polyscience Inc.; Warrington, PA), and the WGA-bindable (WGA+) cells were sorted using a FACScan (Becton Dickinson; San José, CA). TCD-BMCs and WGA+ cells were used for BMT.
BMT
Recipient mice were lethally irradiated one day before BMT. The irradiation dose was 9.0 Gy for the male W/BF1 mice (2 mo), and 9.5 Gy for the C3H/HeN mice (2 mo), since the latter are less radiosensitive. The cells were intravenously injected in the following quantities: 1 to 2 x 107 TCD-BMCs and 1 to 2 x 105 WGA+ cells. Some groups simultaneously received donor bones (two femurs and two tibias) from which the BMCs had been flushed out with medium (RPMI-1640) using a 5 ml syringe with a 24 to 22.5 gauge needle. The bones were 40 Gy-irradiated, since the stromal cells were replaced by donor-derived stromal cells, if the bones were not irradiated, as previously described [7, 10]. The bones were cut at both ends and engrafted in the subcutis (or under the renal capsules). All mice were given antibiotics (ceftazidime 7 mg/ml, minocycline hydrochloride 0.3 mg/ml, and sodium fosfomycin 10 mg/ml) and an antifungal drug (fluconazole 0.15 mg/ml) in their drinking water for one week (in the case of injection of TCD-BMCs) or four weeks (in the case of injection of WGA+ cells). The mice that received the WGA+ cells were further subcutaneously injected with antibiotics and the antifungal drug every day for three weeks.
Histological Studies
Proteinuria was checked, and WBC and platelet counts were made of the peripheral blood. The major organs were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The kidneys were frozen using the OCT compound (Tissue-Tek, Miles, Inc.; Elkhart, IN) and used for immunofluorescence studies.
H-2 Typing of Peripheral Mononuclear Cells in Chimeric Mice
Chimerism was determined using a FACScan by staining mononuclear cells in the peripheral blood with anti-H-2 antibodies (Abs). FITC-anti-H-2Kd (030-21F), FITC-anti-H-2Kb (030-11F) and FITC-anti-H-2Kk (030-39F) Abs were purchased from the Meiji Institute of Health Science (Odawara, Japan). The peripheral blood (Ca. 200 µl volume) was obtained from the vein of the tail or orbita using a heparin-coated microtube (RED TIP, Monoject; St. Louis, MO) and immediately diluted with 400 µl 2 mM-EDTA-phosphate-buffered saline (PBS). The diluted blood was fractionated using a density fractionating medium (M-SMF, JIMRO; Takasaki, Japan) to collect the mononuclear cells. These were suspended in 2% FCS-PBS and stained with FITC-anti-H-2K Abs for 30 min on ice, then fixed in 1.0% paraformaldehyde (PFA)-2% FCS-PBS, and analyzed using a FACScan (Becton Dickinson).
Platelet-Associated Abs (PAAs) and Platelet-Bindable Abs (PBAs)
Peripheral blood was collected in a 15 ml tube with up to 5 ml 2 mM-EDTA-PBS. Platelet-rich-diluted plasma was obtained after double centrifugation at 300 g for 5 min at room temperature. The pellets of platelets were obtained by centrifugation at 1,500 g for 15 min at room temperature. The platelets were suspended in 1 mM-EDTA-PBS, washed twice at 7,000 g for 2 min, fixed with 5% PFA-2 mM-EDTA-2% FCS-PBS and used for staining. FITC-goat-antimouse IgG F(ab)2 (Cappel TM Research Products; Durham, NC) or phycoerythrin (PE)-antimouse-
-chain (Becton Dickinson) was added to 1 x 106/50 ml platelets, which were incubated for 30 min at room temperature. After washing twice, the platelets were analyzed using a FACScan. The PBAs were measured using the sera of the chimeric mice. If the platelet count was too low, only the PBAs in their sera were examined. Approximately 50 µl serum containing 2mM EDTA were obtained from the peripheral blood, to which the platelets (1 x 106) from the BALB/c mice were added, incubated for 30 min at room temperature, washed twice and suspended in 50 µl 1 mM-EDTA-2% FCS-PBS. FITC-antimouse Ig or PE-antimouse--chain was then added, and the mixture incubated for 30 min at room temperature. The platelets were then washed twice and analyzed using a FACScan.
Purification of Pluripotent HSCs (P-HSCs)
P-HSCs were purified from mice injected with 5-fluorouracil (5-FU) according to a new method recently established in our laboratory [11]. Briefly, BMCs were collected from mice given 5-FU (150 mg/kg) three days beforehand. Low-density cells were obtained using a Percoll density gradient technique, and mature hemopoietic cells (CD4+, CD8+, B220+, Mac-1+, Gr-1+ and TER 119+ cells) were then removed using magnetic beads (Dynabeads M-450 SH/RT IgG; Oslo, Norway). These cells were labeled with PE-conjugated anti-CD71 Ab (Pharmingen; San Diego, CA) and appropriate FITC-conjugated anti-H-2 Ab (Meiji). CD71– and H-2high cells were sorted using a FACStar.
CFU-S Assays
P-HSCs were purified from young (<2 mo) or old (>4 mo) W/BF1, C3H and B6 mice, as described above. Recipients were lethally irradiated (9.5 Gy) and then injected with 1 x 103 P-HSCs one day later. All mice were maintained under specific pathogen-free conditions. The recipient mice were sacrificed 12 days after BMT. Their spleens were removed and fixed in Bouin's solution. The surface colonies were counted as CFU-S.
Long-Term Culture of P-HSCs on Stromal Cells
A BM stromal cell line (MS-5), established from the C3H/HeN (H-2k) mouse BM by Itoh et al. [12], was cultured in 25-cm2 flasks containing MEM 
-medium supplemented with 10% horse serum and 1 x 10–6 M of hydrocortisone. When the cells became confluent, they were irradiated by 
-ray (20 Gy), and each flask was cocultured with 103 P-HSCs obtained from old (>4 mo) W/BF1 or C57BL/6 mice. At one-week intervals, the number of cobblestone colonies/flask was counted, and the culture medium containing nonadherent cells was removed from each flask and replaced by fresh medium. The number of collected nonadherent cells was counted. P-HSCs from W/BF1 or C57BL/6 mice were also cultured on 20-Gy-irradiated syngeneic BM adherent cells, and their growth was examined using the same schedule as the culture on MS-5.
Each experimental group consisted of more than five mice or five samples, and each experiment was repeated three or more times. Reproducible results were obtained, and representative data are therefore shown in the figures and the table.
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Results
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Induction of Autoimmune Diseases in C3H Mice by Transplantation of Either TCD-BMCs or Partially Purified HSCs Plus Bone Grafts of W/BF1 Mice
Figure 1
shows the survival rate in (W/BF1 
C3H) chimeric mice. When TCD-BMCs were transplanted from W/BF1 mice to C3H mice, the C3H mice began to show proteinuria and thrombocytopenia three months after BMT, as previously described [9]. Antiplatelet Abs such as PAAs and PBAs were also detected in such chimeric mice (data not shown). All the mice died of renal failure by 250 days after BMT.
The next step was to examine whether autoimmune diseases could be transferred into normal mice by the transplantation of partially purified HSCs from W/BF1 mice. Although the C3H mice that had received partially purified HSCs (1 x 105 WGA+ cells) (without bone grafts) from W/BF1 mice died due to graft failure, those that received partially purified HSCs plus bone grafts from W/BF1 mice began to show proteinuria (++) and thrombocytopenia three months after the transplants. H-2 typing revealed that the hematolymphoid cells were donor-derived (data not shown), as previously described [9]. All the mice died of renal failure due to lupus nephritis by 300 days after the transplants. PBAs were detected in the sera of these mice (data not shown), as previously described [9]. We thus succeeded in inducing autoimmune diseases (lupus nephritis and ITP) in normal mice by transplanting either TCD-BMCs or partially purified HSCs with bone grafts from W/BF1 mice.
Prevention of Autoimmune Diseases in W/BF1 Mice by Transplantation of TCD-BMCs but not by Transplantation of Partially Purified HSCs Plus Bone Grafts from Normal Mice
We have previously shown that the transplantation of TCD-BMCs from normal mice leads to the prevention and treatment of autoimmune diseases in W/BF1 mice, and have here confirmed this. However, when partially purified normal HSCs (WGA+ cells) were used for the transplantation, all mice that received 2 x 105 WGA+ cells died within 25 days due to infection or anemia, even if bones were simultaneously engrafted (Fig. 2). The hematolymphoid organs in such mice showed severe atrophy because of a failure in hematopoietic reconstitution, probably due to the killing of normal HSCs by radioresistant cytotoxic T-lymphocytes (CTLs), natural killer (NK) cells, macrophages and K cells, etc. [13, 14]. These findings strongly suggest that abnormal HSCs are more resilient than normal HSCs; abnormal HSCs can survive even in allogeneic microenvironments, whereas nomal HSCs cannot survive without the help of other cells in such allogeneic microenvironments.
Qualitative Differences Between Abnormal and Normal HSCs Both In Vivo and In Vitro
To confirm that abnormal HSCs can survive even in allogeneic microenvironments, we carried out CFU-S assays on day 12 using P-HSCs (Lin–/CD71–/class Ihigh) purified as described in Materials and Methods.
As shown in Figure 3, both (B6 B6) and (C3H C3H) chimeric mice formed high numbers of colonies in the spleen on day 12 (B6 3.55 ± 0.47/spleen, C3H 5.6 ± 2.14), although (B6 C3H) (0.95 ± 0.24) and (C3H B6) (3.33 ± 1.25) chimeras showed markedly reduced CFU-S counts. However, (W/BF1 (O: old) C3H) chimeras showed significantly higher CFU-S counts (8.29 ± 0.87) on day 12 than those in (B6 C3H) or (C3H B6) chimeras, although the P-HSCs of young (<2 mo) W/BF1 mice (before the development of autoimmune diseases) show major histocompatibility complex (MHC) restriction (the P-HSCs of young W/BF1 mice show very low CFU-S counts in the C3H mouse environment).
To further confirm the existence of qualitative differences between abnormal and normal HSCs, we have established an in vitro assay system. P-HSCs were purified, as previously described [11]. When P-HSCs (Lin–/CD71–/class Ihigh) of B6(H-2b) mice were cocultured with B6 BM adherent cells, they showed a good proliferative response (Table 1). In contrast, when the P-HSCs of B6 mice were cocultured with C3H (H-2k) mouse stromal cells, they showed a poor proliferative response. However, the P-HSCs of old (>4 mo) W/BF1 mice showed no MHC restriction: they could proliferate in collaboration with either W/BF1 or C3H stromal cells. This was the case when P-HSCs were cocultured with a BM stromal cell line, the MS-5 (H-2k). As shown in Figure 4, the P-HSCs of B6 mice showed a poor proliferative response in collaboration with MS-5. However, abnormal P-HSCs of W/BF1 mice showed a much greater proliferative response in collaboration with MHC-incompatible MS-5 cells than when B6 HSCs were cocultured with B6 BM adherent cells (Fig. 4). This was the case when P-HSCs of other autoimmune-prone mice such as BXSB and MRL/lpr mice were cocultured with BM adherent cells (data not shown). We therefore conclude that abnormal P-HSCs are more resilient than normal P-HSCs both in vivo and in vitro.
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Discussion
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It had been thought that the etiopathogenesis of autoimmune diseases could be attributed to abnormalities in T cells (thymus), B cells, macrophages and environmental factors (hormonal disturbances, viral infection, etc.) [15]. However, there has recently been an increase in information suggesting that the abnormalities originate from defects at the HSC level [1-7, 9, 10, 16-20]. In humans, it has also been reported that BMT can be used to treat various autoimmune diseases such as RA and ulcerative colitis, and that autoimmune diseases such as ITP and myasthenia gravis are also transferred to recipients after BMT [21]. Based on these findings, we have proposed that autoimmune diseases are stem cell disorders [22].
In the present study, we confirmed this hypothesis using further purified HSCs, and found a distinct qualitative difference between normal and abnormal HSCs. The transplantation of partially purified HSCs (WGA+ cells) plus bone from W/BF1 mice leads to the development of autoimmune diseases (lupus nephritis, ITP, etc.) in normal mice. As it is difficult to obtain > 1 x 106 WGA+ cells using a FACStar, we transplanted 1-2 x 105 WGA+ cells/mouse with 40 Gy-irradiated bones; the stromal cells in the bones protect HSCs (WGA+ cells) from attack by host cells but do not proliferate. Therefore, we believe that stromal cells from autoimmune-prone mice are not involved in the development of autoimmune diseases, since TCD-BMCs (without bone grafts) from autoimmune-prone mice induce autoimmune diseases in normal mice. The survival rate of the mice that received WGA+ cells plus bone grafts was similar to that of the mice that received TCD-BMCs (Fig. 1
). In contrast, when partially purified HSCs plus bones from normal C3H/HeN mice were transplanted into young W/BF1 mice (<2 mo), the W/BF1 mice died of infection or anemia within three weeks (Fig. 2). Thus, it seems likely that abnormal HSCs are more "resilient" than normal HSCs.
We have recently found that stromal cells present in the BM produce an HSC-chemotactic factor [23], and that an MHC restriction exists between HSCs and stromal cells [7, 24]. Therefore, when HSCs are intravenously injected, the HSCs home in on the engrafted MHC-compatible bones. In vitro analyses of the interaction between the HSCs and stromal cells using a videotape revealed that stromal cells embrace the HSCs (pseudoemperipolesis) (Ogata et al., manuscript in preparation) as if to protect them from exogenous stimuli, including attack by CTLs, NK cells, macrophages, K cells, etc. Thus, bone grafts to recruit donor-derived stromal cells are essential for successful allogeneic BMT. Based on these findings, we transplanted WGA+ cells with bones. However, as shown in Figure 2, the simultaneous transplantation of WGA+ cells and engraftment of bones did not improve the survival rate. Since it is well known that HSCs express considerable quantities of MHC class I, it is conceivable that HSCs are easily killed by radioresistant CTLs, NK cells, macrophages, K cells, etc., before reaching the engrafted bone.
To provide further evidence that abnormal HSCs are more "resilient" than normal HSCs, we performed mixed chimeric experiments. Autoimmune diseases developed in [(autoimmune-prone mice + normal mice) autoimmune-prone mice] chimeric mice, even if normal TCD-BMCs (1 x 107) mixed with abnormal TCD-BMCs (1 x 106) were transferred into the lethally irradiated autoimmune-prone mice [25].
In the present study, we carried out CFU-S assays to confirm that abnormal P-HSCs can survive even in allogeneic microenvironments. As shown in Figure 3, (W/BF1 C3H) chimeric mice showed higher CFU-S counts on day 12 than those in normal allogeneic combinations such as (B6 C3H) and (C3H B6) chimeric mice. In addition, we have found in vitro that further P-HSCs from autoimmune-prone W/BF1 mice proliferate far more rapidly than those from normal mice (Fig. 4 and Table 1). This was the case when P-HSCs of other autoimmune-prone mice such as BXSB and MRL/lpr mice were used (data not shown). Thus, we conclude that abnormal HSCs are indeed more "resilient" than normal HSCs.
We are in the process of clarifying the qualitative differences between normal and abnormal HSCs at the molecular level.
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Acknowledgments
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Supported in part by a grant for Experimental Models for Intractable Diseases from the Ministry of Health and Welfare of Japan, and grants-in-aid for scientific research (02152117), (02670162), (03454177) from the Japan Ministry of Education, Science and Culture.
The authors thank Ms. Y. Shinno and Ms. Y. Matsui for expert technical assistance and Ms. K. Ando for manuscript preparation. We also thank Ms. M. Kataoka and Mr. F. Ishida for conducting FACS sorting.
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References
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Received May 28, 1996;
accepted for publication August 13, 1996.
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Abstract
Introduction
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