Induction of Senile Osteoporosis in Normal Mice by Intra-Bone Marrow-Bone Marrow - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:54 |只看该作者 |倒序浏览 |打印

作者：Yusuke Uedaa,b, Muneo Inabaa,c,d, Keizo Takadaa,b, Junichi Fukuia, Yutaku Sakaguchia, Masanobu Tsudaa, Mariko Omaea, Taketoshi Kushidab, Hirokazu Iidab,c,d, Susumu Ikeharaa,c,d

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      【摘要】, B7 m  w# j( f0 \% [) e9 J, y) |
   A P6 substrain of the senescence accelerated mouse (SAMP6) spontaneously develops osteoporosis early in life. These mice show the clinical signs of osteoporosis, such as elevated levels of urinary deoxypyridinoline (Dpd), decreased bone mineral density (BMD), and a significant loss of trabecular and cortical bone thickness at 12 months of age. Here, we describe the transfer of osteoporosis to a normal strain by the injection of bone marrow cells from SAMP6 donors directly into the bone marrow cavity (intra-bone marrow-bone marrow transplantation , osteoprotegerin, macrophage¨Ccolony-stimulating factor, and insulin-like growth factor-1) were examined by reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR analysis, IL-6 and IL-11 were reduced to a level similar to that in SAMP6 mice, whereas that of RANKL was increased. These findings indicate that not only the hemopoietic system but also the bone marrow microenvironment are reconstituted as a result of IBM-BMT, and suggest that the development of senile osteoporosis might be attributable to "stem cell disorders.": a, K4 p: I9 F, M

Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Bone marrow transplantation Intra-bone marrow injection Senile osteoporosis Senescence accelerated mouse P Stem cell disorder1 V+ l0 }0 a+ Z8 L' k
   INTRODUCTION

Osteoporosis is characterized by progressive loss of bone density, thinning of bone tissue, and increased vulnerability to fractures. For women, bone loss is fastest in the first few years after menopause and continues into the postmenopausal years, and osteoporosis is therefore a major public health concern, it being estimated that 35% of women >65 years old suffer from primary osteoporosis  and might be involved in the pathogenesis of osteoporosis.' k% l) z7 b; V3 s$ ]
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Recently, we have developed a new and effective method for bone marrow transplantation (BMT). Bone marrow cells (BMCs) are directly injected into the bone marrow cavity (the tibia) of recipient mice so that donor-derived hemopoietic cells accumulate in a microenvironment rich in stromal cells . In line with these studies, we attempted to transfer osteoporosis by IBM-BMT from osteoporosis-prone SAMP6 to confirm that osteoporosis is due to "stem cell disorders" in both hemopoietic and mesenchymal lineages.9 E3 k0 K( L0 v3 F# [0 b
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MATERIALS AND METHODS
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Mice5 q$ S" Q$ X5 g0 c5 _# ?# X; Z
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Female SAMP6/Ta (SAMP6, H-2d) were purchased from SLC (Shizuoka, Japan, http://www.jslc.co.jp) and also kindly donated by the Council for SAM Research (Kyoto, Japan, http://samrc.md.shinshu-u.ac.jp/firste.html). The mice were maintained in our animal facility under specific pathogen-free conditions. Female C57BL/6 (B6, H-2b) and C3H/HeN mice (C3H, H-2k) were purchased from SLC. Those mice were maintained until use in our animal facilities under specific pathogen-free conditions.
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Preparation and Inoculation of Bone Marrow Cells. m# G+ U  w+ r( H% ^
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BMCs were collected from the femurs and tibias of SAMP6 at 4 months of age. The whole BMCs were directly injected into the bone marrow cavity (IBM-BMT) of the tibia of B6 mice at 2 months of age. IBM-BMT was carried out according to the method described previously . In brief, the knee was flexed to 90 degrees and the proximal side of the tibia was drawn to the anterior. A 27-gauge needle was inserted into the joint surface of the tibia through the patellar tendon and then inserted into the bone marrow cavity. After removal of the guide, the donor BMCs (3 x 107/10 µl) were injected from said bone hole into the bone marrow cavity using a microsyringe (50 µl; Hamilton Co., Reno, NV, http://www.hamiltoncompany.com).

Experimental Protocols( _3 k2 \0 d8 F" p0 d
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B6 mice (2 months of age) received fractionated irradiation (5.5 Gy x 2; 4-hour interval), and 1 day after the irradiation the mice were transplanted with whole BMCs (3 x 107) from SAMP6 via IBM-injection (IBM-BMT) ().
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Surface Marker Analyses" `; {- }5 h0 L: D
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Spleen cells were prepared from the recipient mice. To distinguish the cells of donor or recipient origin, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-H-2d and phycoerythrin (PE)-conjugated anti-H-2b monoclonal antibodies (mAbs) (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml). FITC- or PE-conjugated mAbs against CD45R (B220), CD4, CD8, CD11b, Gr-1, and RANKL (BD Pharmingen) were used to analyze the cells with mature lineage markers. The cells were analyzed using a FACScan (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).
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Histological Findings2 T, W4 Y( T4 o. z+ ^

The lumbar spine of recipient mice was removed and fixed in 10% formalin and then decalcified. The sections were stained with hematoxylin and eosin (H&E), and osteoclasts were identified by tartrate-resistant acid phosphatase (TRAP) staining.
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Microdensitometry

Bone mass in the femur was roentgenologically assayed according to the method described in our previous paper , and statistical analyses of the bone mass of recipient mice were performed using Student's t test.
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Deoxypyridinoline Analyses
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Urine specimens were collected from the treated and nontreated SAMP6 and B6 mice, and urinary deoxypyridinoline (Dpd) was quantified by an ELISA kit (Metra Biosystems, Inc., Mountain View, CA, http://www.metrabio.com) to evaluate the bone loss. Dpd in urine specimens from female human volunteers of various ages was also measured and used as a control for an ELISA kit, and statistical analyses of urinary Dpd of recipient mice were performed using Student's t test.
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Cultured Stromal Cells
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Cultured stromal cells were obtained as previously described , followed by PE-anti-Rat IgG (Boehringer Mannheim, Mannheim, Germany, http://www.boehringer.com). After blocking with normal rat IgG (BD Pharmingen), the cells were further stained with FITC-anti-H-2d or anti-H-2b and analyzed by a FACScan. The cultured cells stained with isotype-matched Igs served as a negative control.0 t. G$ t1 I' K, i

In Vitro Osteocyte Differentiation Assay* Z0 n. a  J5 |
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Osteogenic differentiation was induced by culturing stromal cells for 3 weeks in differentiation medium: 10% fetal bovine serum in Dulbecco's modified Eagle's medium supplemented with 50 µg/ml ascorbic acid (Sigma-Aldrich), 10 mM ¦Â-glycerophosphate (Sigma-Aldrich), and 0.01 µM dexamethasone (Sigma-Aldrich). The medium was refreshed every 2 days. Mineralized deposits specific for osteocytes were visualized by von Kossa staining. Furthermore, osteoblasts differentiated in this condition were determined by alkaline-phosphatase staining and by the staining with rabbit antiosteocalcin mAb (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) plus FITC-anti-rabbit IgG (Boehringer Mannheim).

Mixed Leukocyte Reaction

Mixed leukocyte reaction (MLR) was performed as follows: The splenic T cells (2 x 105) were cultured with 2 x 105 responder T cells and 2 x 105 irradiated (12 Gy) stimulator spleen cells for 72 hours and pulsed with 0.5 µCi of -thymidine for the last 16 hours of the culturing period.

Reverse Transcription-Polymerase Chain Reaction Assay+ K. m$ C* ^$ q) e4 n8 w$ V

Polymerase chain reaction (PCR) was performed using a mixture of equivalent amounts of cDNA of each sample, Gene Taq, 10 x Gene Taq universal buffer, dNTP Mixture (Nippon Gene, Tokyo, http://www.nippongene.com), each of gene-specific primer sets for IL-11, IL-6, RANKL, osteoprotegerin (OPG), macrophage-colony-stimulating factor (M-CSF), and insulin-like growth factor (IGF)-1, and control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The sequences of the primer sets are shown in Table 1. All PCRs were performed by a TaKaRa PCR Thermal Cycler Personal (Takara, Otsu, Japan, http://www.takara.co.jp). The cycling conditions comprised a denaturation step for 5 minutes at 94¡ãC followed by 40 cycles of denaturation (94¡ãC for 30 seconds), annealing (60¡ãC for 30 seconds), and extension (72¡ãC for 30 seconds). The PCR products were electrophoresed on a 2% agarose gel, stained with ethidium bromide (0.5 µg/ml), and visualized by an UV transilluminator.
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Table 1. Sequence of primer sets in real-time reverse transcription-polymerase chain reaction assay% j2 k$ i' W! B6 V" W
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Real-Time Reverse Transcription-PCR Assay

Real-time reverse transcription (RT)-PCR was conducted on a DNA Engine Opticon 2 System (MJ Japan Ltd., Tokyo, http://www.biocompare.com) by using SYBR Green I as a double-stranded DNA-specific binding dye and continuous fluorescence monitoring. The cycling conditions comprised a denaturation step for 10 minutes at 95¡ãC followed by 40 cycles of denaturation (94¡ãC for 15 seconds), annealing (60¡ãC for 30 seconds), and extension (72¡ãC for 30 seconds). After amplification, melting curve analysis was performed with denaturation at 95¡ãC then continuous fluorescence measurement from 65¡ãC to 95¡ãC at 0.1¡ãC/second. Primers for Il-11, IL-6, RANKL, and control GAPDH were the same as those used for RT-PCR. All reactions were run in duplicate, at least, and included control wells without cDNA.- h$ g; [, w! ^6 z2 t

RESULTS/ b' e: @1 u9 w3 E1 b) ]! W  e
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We have previously reported that BMT via IBM-BMT facilitates early donor cell engraftment, resulting in the reconstitution of not only hemopoietic cells but also stromal cells of donor origin. Using this method, we have succeeded in preventing and treating osteoporosis in SAMP6. To determine whether the development of osteoporosis is attributable to the "stem cell," we attempted to transfer osteoporosis to normal mice by IBM-BMT from SAMP6. The following analyses were carried out after IBM-BMT.. F6 O' U  Z' \5 h- I9 g
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Cell Surface Antigens( g' a) v0 T+ ^7 d; D4 @

We carried out fluorocytometrical analyses using cells harvested from the recipient B6 mice and examined the engraftment of donor-derived cells and immunological functions. The percentage of donor (SAMP6)-derived cells (H-2d ) in the spleen of  and nontreated SAMP6 mice, indicating that the activation of osteoclastogenesis by the systemic increase of RANKL.

Table 2. Cell surface antigens on donor-derived cells in spleens of  mice

Next, we examined the reconstitution of stromal cells that have been reported to be of mesenchymal origin and participate in bone formation. Cultured stromal cells were double-stained with stromal-cell-specific mAb (anti-PA6 mAb)  mice were confirmed to be of donor origin (H-2d ), indicating that not only cells of hemopoietic lineage but also those of mesenchymal origin were reconstituted with donor-type cells after IBM-BMT. It is noted that the reconstitution of stromal cells by the cells of donor origin was observed only when IBM-BMT had been performed, not when IV-BMT was performed. After IV-BMT, both donor- and recipient-derived stromal cells were detected in the 3-week culture (data not shown).3 v$ a1 n& w) x4 f
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Figure 1. Analysis of stromal cells from B6 mice treated with IBM-BMT. The bone pieces without bone marrow cells from B6 mice treated with IBM-BMT were cultured for 3 weeks, and then the adherent cells were collected. (A): The adherent cells were stained with anti-PA6 monoclonal antibody (mAb) followed by phycoerythrin (PE)-anti-Rat IgG then blocked with normal rat IgG. They were further stained with fluorescein isothiocyanate (FITC)-anti-H-2d mAb (donor type) or FITC-anti-H-2b mAb (recipient type). In the mice treated with IBM-BMT (left panels), bone marrow stromal cells were completely reconstituted with donor-derived cells, whereas those from the recipients treated with IV-BMT (right panels) show the profile of mixed chimerism. The profile of the cells stained with FITC-anti-H-2b mAb is similar to that stained with an isotype-matched Ig control (data not shown). (B): Stromal cells were cultured for 3 weeks in osteogenic differentiation medium, and osteoblasts differentiated in this condition were determined by the staining with rabbit antiosteocalcin mAb plus FITC-anti-rabbit IgG. Donor-derived cells were confirmed by PE-anti-H-2d mAb. A merged image is shown. The stained samples were examined on a confocal laser scanning microscope (FV300; Olympus, Tokyo, http://www.olympus-global.com) equipped with a x20 objective lens. Abbreviations: IBM-BMT, intra-bone marrow-bone marrow transplantation; IV-BMT, intravenous-bone marrow transplantation.

Furthermore, osteocytes, defined by von Kossa staining and alkaline-phosphatase staining, were differentiated from the stromal cells that were of donor origin when the stromal cells were cultured for a further 3 weeks in the presence of differentiation medium (data not shown). The origin of osteocytes differentiated in vitro was confirmed by double-staining with anti-H-2d and antiosteocalcin mAb, as shown in Figure 1B, where osteocalcin-positive cells were also positive for donor-type H-2d. These findings strongly suggest that the osteocytes in the recipients were also of donor origin, and these donor (SAMP6)-derived abnormal osteocytes might contribute to development of senile osteoporosis in the recipient B6 mice.

Immunological Functions) j, x9 Z& O/ F! F

Newly developed T cells showed tolerance to both host (B6)-type and donor (SAM6)-type major histocompatibility complex determinants, whereas they showed normal responses to third party (C3H) cells when examined in MLR (Fig. 2A). These findings indicate that newly developed T cells are immunologically competent and that self-tolerance is induced and maintained in the recipients after IBM-BMT.& G" s. K; u6 j+ C" k
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Figure 2. Analyses for the function of T cells in B6 mouse treated with intra-bone marrow-bone marrow transplantation (IBM-BMT). (A): Mixed leukocyte reaction. The splenic responder T cells (2 x 105) were cultured with 2 x 105 irradiated (12 Gy) stimulator spleen cells for 72 hours and pulsed with 0.5 µCi of -thymidine for the last 16 hours of the culture period. *, p ' j% I  x& P  N+ {2 g# E+ p1 s

Histopathological Findings" y1 Q% l& t2 b# x! ^

The aged SAMP6 exhibited histopathological findings of osteoporosis. Figure 3A¨C3E shows the histology of a lumbar spinal vertebral body. SAMP6 showed a significant loss of trabecular and cortical bone thickness at 8 months of age (Fig. 3C) when compared with B6 mice of the same age (Fig. 3A). Furthermore, a large number of adipocytes were observed in the specimens from SAMP6. It is noted that B6 mice treated with IBM-BMT from SAMP6 () (Fig. 3B), suggesting that irradiation itself was not a participant in the development of osteoporosis.

Figure 3. Histological findings of the lumbar spinal vertebral body and changes in bone mineral density (BMD). The lumbar spinal vertebral body of B6 (A) or SAMP6 (C) at 8 months of age. Significant loss of trabecular bone, cortical bone thickness, and distribution of adipocytes was observed in the SAMP6 specimen (C) but not in the B6 specimen (A). The B6 mouse that received bone marrow cells (BMCs) from the SAMP6 by IBM-BMT ( mouse using IV-BMT (G). Abbreviations: GS/D, area of bone pattern/bone width; IBM-BMT, intra-bone marrow-bone marrow transplantation; IV-BMT, intravenous-bone marrow transplantation; SAMP6, P6 substrain of the senescence accelerated mouse.

Bone Mineral Density

As shown in Figure 3F, normal B6 mouse showed the highest bone mineral density (BMD) at 12 months of age, and this gradually decreased, whereas the highest BMD was observed at 5¨C6 months of age in SAMP6 and thereafter rapidly decreased. After the treatment of B6 mice at 2 months of age with IBM-BMT (), the BMD was similar to or lower than that of SAM6. And at 12 months and 20 months, decreases in BMD were confirmed (Fig. 3G) as observed in the abovementioned histopathological findings.

Urinary Deoxypyridinoline5 u- X3 N! x6 Y8 Q5 l, V' v

An increase in urinary Dpd is one of the clinical hallmarks of bone resorption. Actually, Dpd in SAMP6 rapidly increased after 6 months of age, in contrast to that in normal B6 mice (Fig. 4A), indicating that osteoclastogenesis in SAMP6 is activated. Furthermore, Dpd in the  mice at 12 and 20 months. These findings again suggest that the reconstitution of both HSCs and MSCs caused activated osteoclastogenesis to contribute to development of senile osteoporosis in recipient B6 mice.% u: H# m+ j7 n
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Figure 4. Kinetic changes of urinary deoxypyridinoline. Dpd was measured by ELISA, and Dpd was increased in B6 mice treated with IBM-BMT from SAMP6 (blanked triangle) compared with those treated with IBM-BMT from B6 mice (blank circle). Square indicates control SAMP6, and diamond indicates control B6 mice. Symbols represent the means of five mice (A). The analyses were performed using Student's t test at the time points of 4 months, 12 months, and 20 months after birth. *, p

TRAP Stain! m3 P3 R( a8 z' C% g! i. Q

To confirm increased urinary Dpd due to activated osteoclastogenesis, we investigated the activity of osteoclasts in the lumbar spinal vertebral body by TRAP staining. In the  mice (12 months) (Fig. 4C¨C4F), suggesting that activated osteoclastogenesis facilitates the increases in bone resorption.% H8 T* O2 J, e8 o( M' i0 I8 G. V( `
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Analyses of Cytokines
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We next investigated the cytokines produced from these cells and their involvement in the bone formation. Several cytokines, including IL-6 , are related to bone formation or resorption. Thus, we next examined some of these cytokines at the message level by RT-PCR and/or real-time RT-PCR in bone marrow stromal cells.% u. V3 P% x! q- |( n

Stromal cells were collected from the B6 mice treated with IBM-BMT from SAMP6 and control mice (age-matched, nontreated B6 mice or SAMP6), and the message levels of IL-6, IL-11, M-CSF, OPG, RANKL, and IGF-1 were compared. RANKL that activates osteoclast progenitors to differentiate into mature osteoclasts was higher in the  mice. In the other cytokines related to the bone formation or remodeling such as M-CSF, OPG, and IGF-1, no significant changes were observed (Fig. 5A).9 d. |; V9 b5 |; Y& H

Figure 5. Analyses of cytokine messages. The expression of mRNA of IL-11, IL-6, M-CSF, and IGF-1 in stromal cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) (A). To confirm the significance in the message level of IL-11 and IL-6, real-time quantitative RT-PCR was performed. Relative intensity of IL-11 (B) or IL-6 mRNA (C) was calculated on the basis of GAPDH intensity. Data are shown as mean ¡À SD of five mice. *, p 5 n) c  U# ^1 k. O4 ]  M
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DISCUSSION( g9 K1 k, ^  }) L

We have previously carried out allogeneic BMT studies using conventional IV-BMT and IBM-BMT in various mouse combinations such as  combination as a control for irradiation.
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We have recently shown that not only the hemopoietic cells but also the stromal cells that constitute the bone marrow microenvironment are completely reconstituted by donor-derived cells after IBM-BMT, resulting in the prevention and treatment of osteoporosis through an amelioration of the imbalance between bone resorption and formation  mice showed decreased trabecular bone and bone mineral density (BMD) but increased urinary deoxypyridinoline (Dpd), a hallmark of bone resorption. The recipients of IBM-BMT had a hematolymphoid system of donor origin, and the osteocytes that had differentiated from stromal cells were also of donor origin, as confirmed by the double staining with anti-H-2d and antiosteocalcin. Thus, osteoporosis could be transferred to normal recipients by IBM-BMT, which allowed not only HSCs but also MSCs to be replaced.

When the message levels of cytokines (IL-6, IL-11, RANKL, M-CSF, OPG, and IGF-1) were examined, the expressions of IL-11 and IL-6 decreased in the recipients of IBM-BMT from SAMP6 ( mice was thus similar to those in SAMP6, indicating the induction of osteoporosis.% C  v. t0 D5 K, a2 n

It has been widely accepted that RANKL, RANK, and OPG are essential for controlling the osteoclast development and functions in bone remodeling, and the inhibition of RANKL activity by OPG injections results in significantly reduced bone loss in arthritis  mice was clearly observed, the frequency of these cells being similar to that in untreated SAMP6 mice. This might explain the development of senile osteoporosis after the activation of osteoclastogenesis by a systemic increase of RANKL; probably soluble RANKL was released from CD4  T cells with increased RANKL expression.8 |8 a1 j2 V( R

In the present study, we have shown that HSCs and MSCs were reconstituted with cells of donor origin in the recipients treated with IBM-BMT (Fig. 1A). However, IV-BMT failed to either transfer or treat osteoporosis from SAMP6 to normal mice or vice versa (Fig. 3D, 3F, 3G), since IV-BMT can replace the recipients with donor-derived hemopoietic cells but not stromal cells (Fig. 1A). These findings strongly suggest that the development of senile osteoporosis is due to an abnormality in the mesenchymal stem cells.( S5 T5 Z' A8 ]) W0 q, y+ ^
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To further examine the effectiveness of IBM-BMT to reconstitute not only HSC-lineage cells but also MSC-lineage cells, we attempted to transfer osteoporosis to normal recipients and actually succeeded in developing this disease after IBM-BMT in conjunction with the cytokine pattern observed in the osteoporosis-prone SAMP6. This is in accordance with our previous data that MSC-derived stromal cells are crucial in the development of autoimmune disease; we showed that the cotransplantation of stromal cells and BMCs from autoimmune-prone MRL/lpr mice can induce systemic autoimmune diseases in normal B6 recipients .

The most widely accepted protocol after the onset of osteoporosis is combination therapy consisting of pharmacological and dietary interventions. However, these protocols have recently been questioned due to the risk/benefit ratio of prolonged treatment. Thus, there is a critical need for safe and effective alternative therapeutics for this disease. From this standpoint, we have shown in our series of experiments that IBM-BMT can modify both HSC-lineage and MSC-lineage cells, resulting in the treatment or transfer of the disease. Therefore, it can be concluded that IBM-BMT can be also applied to the treatment of senile osteoporosis.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.7 u: f8 y( Z. _8 I

ACKNOWLEDGMENTS( v2 c' ~: k7 \' y4 i% j

We thank Y. Tokuyama, A. Kitajima, and K. Hayashi for their expert technical assistance and Hilary Eastwick-Field and K. Ando for their help in the preparation of the manuscript. This work was supported by a grant from the Haiteku Research Center of the Ministry of Education; a grant from the Millennium program, the Science Frontier program, and the 21st Century Center of Excellence (COE) program of the Ministry of Education, Culture, Sports, Science and Technology; a Grant-in-Aid for scientific research (B) 11470062; Grants-in-Aid for scientific research on priority areas (A) 10181225 and (A) 11162221; Health and Labour Sciences research grants (Research on Human Genome, Tissue Engineering Food Biotechnology); a grant from the Department of Transplantation for Regeneration Therapy (sponsored by Otsuka Pharmaceutical Company, Ltd.); a grant from Molecular Medical Science Institute, Otsuka Pharmaceutical Co., Ltd.; and a grant from Japan Immunoresearch Laboratories Co., Ltd. (JIMRO).8 m1 ~  N- _1 B2 e4 w6 C
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