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THE STEM CELL NICHE

Analyses of Very Early Hemopoietic Regeneration After Bone Marrow Transplantation: Comparison of Intravenous and Intrabone Marrow Routes

^aFirst Department of Pathology,
^bRegeneration Therapy,
^cRegeneration Research Center for Intractable Diseases, Kansai Medical University, Osaka, Japan;
^dDepartment of Pathology, North Taiping Road Hospital, Beijing, China

Key Words. Intravenous-bone marrow transplantation • Intrabone marrow-bone marrow transplantation • Hemopoietic regeneration Chemotaxis

Correspondence: Susumu Ikehara, M.D., Ph.D., First Department of Pathology, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi City, Osaka 570-8506, Japan. Telephone: 81-6-6993-949; Fax: 81-6-6994-8283; e-mail: ikehara{at}takii.kmu.ac.jp

Received on June 9, 2006; accepted for publication on January 3, 2007.

First published online in STEM CELLS EXPRESS  February 22, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
In bone marrow transplantation (BMT), bone marrow cells (BMCs) have traditionally been injected intravenously. However, remarkable advantages of BMT via the intra-bone-marrow (IBM) route (IBM-BMT) over the intravenous route (IV-BMT) have been recently documented by several laboratories. To clarify the mechanisms underlying these advantages, we analyzed the kinetics of hemopoietic regeneration after IBM-BMT or IV-BMT in normal strains of mice. At the site of the direct injection of BMCs, significantly higher numbers of donor-derived cells in total and of c-kit⁺ cells were observed at 2 through 6 days after IBM-BMT. In parallel, significantly higher numbers of colony-forming units in spleen were obtained from the site of BMC injection. During this early period, higher accumulations of both hemopoietic cells and stromal cells were observed at the site of BMC injection by the IBM-BMT route. The production of chemotactic factors, which can promote the migration of a BM stromal cell line, was observed in BMCs obtained from irradiated mice as early as 4 hours after irradiation, and the production lasted for at least 4 days. In contrast, sera collected from the irradiated mice showed no chemotactic activity, indicating that donor BM stromal cells that entered systemic circulation cannot home effectively into recipient bone cavity. These results strongly suggest that the concomitant regeneration of microenvironmental and hemopoietic compartments in the marrow (direct interaction between them at the site of injection) contributes to the advantages of IBM-BMT over IV-BMT.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
In bone marrow transplantation (BMT), bone marrow cells (BMCs) are traditionally transfused intravenously into systemic circulation. After they escape the specific and/or nonspecific trapping in the lung, liver, spleen, etc [1–3], some of the bone marrow (BM) progenitor cells can home to the BM spaces, followed by the regeneration of the hemopoietic system [3–7]. However, the seeding efficiency of BMCs into the BM tissue is known to be very low [3–7]. In addition, there is no direct evidence that hemopoietic stem cells (HSCs) that contribute to re-establishing and sustaining the recipient hemopoietic system over the long term are ready to home to their physiological sites. Some stem cells in the "niches" might not express the relevant homing receptors, as is the case with CD34^– hemopoietic stem cells in humans [4].

To improve the outcome of BMT, therefore, it is reasonable to deliver BMCs directly into the BM. In fact, we and others have documented the remarkable advantages of intrabone-marrow (IBM)-BMT over intravenous (IV)-BMT in refractory BMT settings, such as [normalMRL/lpr mice] [8] and [human BMCsSCID mice] [3, 4, 9]. Moreover, we have found recently that senile osteoporosis in SAMP6 mice can be prevented and treated effectively by IBM-BMT using BMC from healthy mice [10, 11]. In the recipient mice, the proliferation of donor stromal cells was also observed at the site of injection [8, 11]. These results indicate the possibility that the stromal cells contained in donor BMCs play an important role in IBM-BMT. To investigate the role of stromal cells precisely, we performed the simultaneous injection of donor BMCs and a stromal cell line (PA6 cells) into recipient bone cavity in a normal mouse combination [12]. As expected, higher white blood cell, red blood cell, and platelet counts and colony-forming units in spleen (CFU-S) formation were observed only 12 days after BMT in the mice that received IBM injection of BMCs plus PA6 cells than in those that received IV injection of BMCs plus PA6 cells or IBM injection of BMCs alone. Thereby, the IBM-BMT group showed the highest survival rate of the three groups up to 60 days after BMT. When allogeneic BM-adherent cells were used instead of the PA6 cells, a similar enhancement in CFU-S formation was observed in the IBM-BMT group [12]. Thus, it is conceivable that stromal cells provide an advantage for hemopoiesis when injected directly into bone cavity.

These results indicated that IBM-BMT is more effective than IV-BMT. However, what happens at the site of injection of BMCs in the early phase of regeneration and how the direct injection of BMCs (including stromal cells) contributes to the advantages of IBM-BMT remain to be clarified. In the current study, we investigated the kinetics of hemopoietic regeneration, not only at the site of injection but also at the other sites. We here show a clear difference in the early phase of hemopoietic regeneration between the BM site injected with donor BMCs and other BM sites injected with saline alone in IV-BMT or not injected with donor BMCs in IBM-BMT. Histological studies also show the earlier accumulation/proliferation of both hemopoietic cells and stromal cells only when the BM is directly injected with donor BMCs. Thus, these results suggest that the direct injection of BMCs into bone cavity induces the earlier recovery of the BM microenvironment and that this process is very important for the earlier regeneration of hemopoietic cells. We also show that, when stromal cells enter into systemic circulation by IV-BMT, they cannot migrate effectively into bone cavity.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
Mice
Seven week-old female B10.A (H-2^a), B10.BR (H-2^k), BALB/c (H-2^d), and B57BL/6 (H-2^b) mice were purchased from Shizuoka Experimental Animal Co. Ltd. (Hamamatsu, Japan) and maintained under pathogen-free conditions in our animal facility throughout the study. All mice were kept for at least 2 weeks before the initiation of the experiments. The university's committee for animal research approved all experiments.

Whole-Body Irradiation of Mice
Gamma-irradiation was delivered by a Gammacell 40 Exactor (MDS Nordion, Kanata, ON, Canada, http://www.mds.nordion.com/) with two ¹³⁷Cs sources at the dose rate of 1.05 Gy/minute. Recipient mice were lethally irradiated (9.5 Gy) 16–18 hours before BMT. In some experiments, mice were irradiated (9.5 Gy) to get their sera and BMCs at various time points after irradiation.

Isolation of BMCs
BMCs were flushed from the medullary cavities of the humeri, femora, and tibiae of donor mice, using a 26-G needle attached to a 1-ml syringe, into phosphate-buffered saline with 2% fetal calf serum. After gentle dissociation, the BMC suspension was filtered through cotton mesh, and BMCs were counted with a hemocytometer or by an electronic counter (Sysmex K-1000; Sysmex, Kobe, Japan, http://www.sysmex.co.jp). The erythrocyte counts in the BMC suspension were very low because the mice were killed by exsanguination.

Bone Marrow Transplantation
The day after irradiation, the BMCs were transplanted into recipient mice directly into the bone cavity (IBM-BMT) or intravenously (IV-BMT). For IBM-BMT, the mice were anesthetized with pentobarbital sodium (0.05 g/g of b.wt.), and the left tibia was gently drilled with a 26-G needle through the patellar tendon. The BMCs (1 x 10⁷ in 10 µl) were injected into the bone cavity through the hole in the tibia using a Hamilton microsyringe.

The IV-BMT mice were injected with the same number of BMCs via a tail vein. In addition, the mice were injected with 10 µl of saline into the left tibiae as a sham operation.

Analyses for Surface Marker Antigens
The recipient mice were sacrificed periodically after IBM- or IV-BMT. The single cells were recovered from humeri, tibiae, femora, and spleens, individually. The cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-H-2D^d monoclonal antibody (mAb) for B10.A mice or phycoerythrin (PE)-conjugated anti-H-2K^d mAb (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) for BALB/c mice to identify the donor-derived cells, and the PE-conjugated anti-H-2D^k mAb for B10.BR mice or FITC-conjugated anti-H-2K^b mAb for C57BL/6 mice to identify the recipient cells. In some experiments, the cell surface phenotypes were analyzed by PE-conjugated erythroid-, myeloid-, and lymphoid-lineage markers (Lin; anti-CD3, anti-CD8, anti-B220, anti-CD11b (Invitrogen, Carlsbad, CA), anti-CD4, anti-Gr-1, anti-TER119 mAbs) and PE-conjugated anti-H-2K^b mAb, FITC-conjugated anti-c-kit mAb and biotinylated anti-CD34 mAb followed by streptavidin-cychrome (BD Pharmingen). The stained cells were analyzed using a FACScan (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) equipped with CellQuest software.

Colony-Forming Unit in Spleen
For CFU-S assays, only C57BL/6 mice were used because of a severe shortage of B10 congenic mice at the animal breeders. The recipient mice of [BALB/cC57BL/6] were sacrificed on days 2, 4, 6, and 9 after BMT. The BMCs were collected from left and right tibiae individually. Single cell suspensions from each tibia were prepared, and the numbers of cells were counted with a hemocytometer. The BMCs (2 x 10⁵/0.2 ml) were intravenously injected into lethally irradiated (9.5 Gy) C57BL/6 mice. Eight days after the injection, the spleens were removed and placed in Bouin's fixative. The numbers of colonies were counted under a dissecting microscope. Histological analyses of CFU-S were also carried out.

Pathological Examination
Mice were sacrificed for pathological examination at 3 and 8 hours, and 1, 2, 4, 6, 9, and 14 days after BMT. The humeri, femora, tibiae, ribs, vertebral bodies, and ilium were removed and fixed in 4% formaldehyde solution. The bones were then decalcified and longitudinally cut into two halves. Thymus, spleen, liver, and mesenteric lymph nodes were also removed, weighed, and fixed. Both halves of bones and other organs were processed for H&E preparations.

Detection of Donor-Type Stromal Cells
BMCs were collected from the tibiae of the recipient mice of [BALB/cC57BL/6] on days 2, 4, and 6 after BMT (5–7 mice/time point) and then cultured in dishes containing 10% fetal bovine serum (FBS)/Iscove's modified Dulbecco's medium (IMDM) for 2 weeks. Adherent cells were recovered from the dishes and double-stained with anti-H-2D^d mAb (donor-type) plus anti-CD45 antibody (BD Pharmingen). H-2D^d-positive and CD45-negative cells were considered donor-type stromal cells.

Collection of Sera and BM Tissue Extracts from the Irradiated Mice
C57BL/6 mice were irradiated (9.5 Gy) and sacrificed for collecting sera and BMCs between 4 hours and 4 days after the irradiation. BM tissue extracts were prepared by incubating BMCs obtained from four legs of normal or irradiated mice in 8 ml of serum-free medium (Stem span; StemCell Technologies, Inc., Vancouver, BC, Canada, http://www.stemcell.com/) for 24 hours in 5% CO₂ in air. The extracts were centrifuged at 3,000 rpm and passed through a 45-µm filter. The collected sera were also centrifuged and filtrated.

Migration Assay of Stromal Cells
Using a 24-well transwell system, sera and BM tissue extracts obtained from irradiated mice were examined to determine whether they contain chemotactic factors capable of promoting the migration of BM stromal cells. One milliliter of 10% FBS/IMDM containing 10% of the sera and BM tissue extracts was placed in the lower chamber. A stromal cell line (FMS/PA6-P [13]: 10⁴ cells) in 0.5 ml of medium was loaded into the upper chamber (cell insert: pore size, 8 µm; chemotaxicell; Kurabo, Osaka, Japan, http://www.bio.kurabo.co.jp/English/index.htm) so that the cells can migrate. The Transwell (six wells per sample) was placed in a CO₂ incubator for 4 days. During the incubation time, the migrating FMS/PA6-P cells adhered to the surface of the 24-well plate and proliferated there, forming colonies of 10–20 cells. Four days later, the cell insert and the medium were removed, and the adherent cell colonies on the 24-well plate were then counted after May-Giemsa staining. A single stromal cell forms one colony; therefore, the number of colonies represents the number of stromal cells to have migrated through the membrane. Each sample was run in triplicate.

In our preliminary experiments, sera or BM tissue extracts were placed in the lower chamber at concentrations of 5–50%, and their chemotactic activity was examined for FMS/PA6-P cells. Maximum migration was observed at the concentrations of 10%–20% of each sample; therefore, sera or BM extracts were used at 10% in all the migration assays thereafter.

Chemotactic activity of recombinant human hepatocyte growth factor (rhHGF), provided by Mitsubishi Pharma Corporation (Osaka, Japan, http://www.m-pharma.co.jp/e/index.php), was also examined in the Transwell assay system. Polyclonal goat anti-human hepatocyte growth factor (HGF) antibody (Techne Corporation, Minneapolis, http://www.techne-corp.com/) was added to the lower wells containing rhHGF or BM tissue extracts to neutralize the chemotactic activity of HGF.

Statistical Analyses
We used more than three mice per time point (3 and 8 hours and 1, 2, 3, 4, 6, 9, 14, 17, and 30 days after IBM-BMT or IV-BMT) and repeated the BMT experiments more than 20 times. Statistical analysis was performed with the use of SPSS 8.0 (SPSS Inc., Chicago, IL, http://www.spss.com/). The means of numbers or percentages of different types of donor-derived hematopoietic cells in BMs or spleens were analyzed by one-way analysis of variance. If the homogeneity of variance of samples was equal, the means of left and right tibiae in IBM- and IV-BMT were compared using least significant difference in a post hoc test. If the homogeneity of variance of samples was not equal, the means of each group in the tibiae were compared with Tamhane's T2 and nonparametric tests. If the homogeneity of variance of sample groups was not equal, the means of groups in tibiae and spleens in IBM-BMT and IV-BMT groups were compared with nonparametric tests. Statistical significant differences in migration assays were analyzed by Student's 2-tailed t tests.

	RESULTS

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Kinetics of Donor-Derived Hemopoiesis
In the first set of experiments, we compared the kinetics of regeneration of bone marrow cells in the [B10.AB10.BR] chimeras. The recipient B10.BR mice were sacrificed at various time points (hour 3, days 1, 3, 6, 9, 17, or 30) after IBM-BMT or IV-BMT. Cells were prepared from the humeri, femora, tibiae, and spleens, double-stained with anti-H-2D^d mAb (donor-type) and anti-H-2D^k mAb (recipient-type), and analyzed by FACScan.

Figure 1 shows the kinetics of hemopoietic regeneration after total body lethal irradiation plus IBM-BMT or IV-BMT. As shown in Figure 1A, the total number of bone marrow cells had decreased drastically by day 3, followed by a rapid increase by day 9 after BMT. Among these BMCs, the donor-derived cells were initially approximately 10% in the BMC-injected tibia, but only approximately 1% were of donor-origin in the contralateral tibia in the IBM-BMT group and in both tibiae in the IV-BMT group (Fig. 1B). The percentages of donor-derived cells steadily increased to near 100% by day 6 in both groups. Reflecting the differences in the percentages, the total numbers of donor-derived cells in each bone were clearly more in the BMC-injected tibiae (Fig. 1C). During this early phase of hemopoietic regeneration, donor-derived cells were detected more and in higher frequency only in the BMC-injected tibiae (p < .001). On day 9 and thereafter, the difference was no longer obvious.

View larger version (40K): [in this window] [in a new window]   Figure 1. Kinetics of hemopoietic regeneration in tibia after IBM-BMT or IV-BMT. (A): Total numbers of bone marrow cells. (B): Percentages of donor-derived cells. (C): Total numbers of donor-derived cells. The percentage and number of donor-derived cells are significantly higher in the BMC-injected tibiae () than the contralateral tibiae () in the IBM-BMT group or the saline-injected () and noninjected () tibiae in the IV-BMT group during the early period after BMT. Abbreviations: BMC, bone marrow cell; BMT, bone marrow transplantation; IBM, intrabone-marrow; IV, intravenous.

Kinetics of "Lymphocyte," "Blast," and "Granulocyte" Window Cells
On a side light scatter (SSC)/forward light scatter (FSC) profile of a fluorescence-activated cell sorting dot plot, the BMCs can be roughly divided into "lymphocyte," "blast," and "granulocyte" window cells (Fig. 2A) [14], where most of the cells in each window are B lymphocytes, immature myeloid progenitor cells, or granulocytes, respectively.

View larger version (35K): [in this window] [in a new window]   Figure 2. Kinetics of the regeneration of donor-derived lymphocytes, blastic cells, and granulocytes. (A): Three windows ("lymphocyte," "blast," and "granulocyte") on SSC/FSC profile. The majority of cells in each of the three windows are B lymphocytes, immature myeloid progenitor cells, and granulocytes, respectively. (B, C): Kinetics of the regeneration of donor-derived cells belonging to the "lymphocyte" (), "blast" (), and "granulocyte" () windows. (B): Bone marrow-injected tibia in the IBM-BMT group. (C): Saline-injected tibia in the IV-BMT group. The donor-derived cells in all three windows at the site of BMC injection started to increase earlier than at the site of saline-injection. Abbreviations: BMC, bone marrow cell; BMT, bone marrow transplantation; FACS, fluorescence-activated cell sorting; FSC, front light scatter; IBM, intrabone-marrow; IV, intravenous; SSC, side light scatter.

Kinetics of Lin^–/CD34⁺ and Lin^–/c-kit⁺ Cells
To trace the hematopoietic precursor cells, donor-derived BMCs were further divided into Lin^–/CD34⁺ (Fig. 3A) and Lin^–/c-kit⁺ cells (Fig. 3B) in [BALB/cC57BL/6] chimeras. As shown in Figure 4A, Lin^–/CD34⁺ donor-derived cells had, to some extent, increased at the site of BMC injection on days 4 and 6 after IBM-BMT (but not significant). However, the differences in the number of donor-derived Lin^–/c-kit⁺ cells in the BM-injected tibiae were statistically significant (p < .01) on days 4 and 6 (Fig. 3B). In the spleens, there were very few donor-derived Lin^–/CD34⁺ and Lin^–/c-kit⁺ cells even on day 9 after BMT, and no difference was observed between the IBM-BMT and IV-BMT groups (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 3. Kinetics of the regeneration of donor-derived Lin–CD34+ (A) and Lin–c-kit+ (B) cells in tibia after IBM-BMT or IV-BMT. The number of donor-derived Lin–c-kit+ cells at the site of BMC-injection () was significantly higher than the contralateral tibiae () in the IBM-BMT group and the saline-injected () and noninjected () tibiae in the IV-BMT group on days 4 and 6 (p < .01). Abbreviations: BMC, bone marrow cell; BMT, bone marrow transplantation; IBM, intrabone-marrow; IV, intravenous.

View larger version (29K): [in this window] [in a new window]   Figure 4. Kinetics of the regeneration of CFU-S. The number of CFU-S in the BM at the site of BMC-injection () was significantly higher than that in the contralateral tibiae in the IBM-BMT group () and both tibiae in the IV-BMT group ( or ) on days 2 (p < .001), 4 (p < .01), and 6 (p < .05). Abbreviations: BMC, bone marrow cell; BMT, bone marrow transplantation; CFU-S, colony-forming units in spleen; IBM, intrabone-marrow; IV, intravenous.

As shown in Figure 4, the number of CFU-S in the BM at the site of BMC-injection was significantly higher than in the contralateral tibiae in the IBM-BMT group: both tibiae in the IV-BMT group on days 2 (p < .001), 4 (p < .01), and 6 (p < .05). Furthermore, the increase in the number of CFU-S began at the BMC-injection site more rapidly than all other sites. However, all other sites showed a delayed but vigorous increase in CFU-S between days 4 and 6 after BMT. On day 9, all tibiae contained similar numbers of CFU-S.

The histological analyses of CFU-S were carried out and compared among various bone sites at each time point. No significant difference was observed among them. However, there was a tendency for the percentage of more immature CFU-S composed of three lineages (erythroid cells, myeloid cells, and megakaryocytes) to be higher at the site of the BMC injection than at other bone sites on the day 4 after IBM-BMT.

Kinetics of Histology
After severe depletion of lymphocytes as early as 1 day after BMT, the thymus and lymph nodes slowly recovered cellularity by days 9–14. In contrast to these lymphoid tissues, spleens showed prominent proliferation of hemopoietic cells on days 6, 9, and 14 after initial depletion of both hemopoietic and lymphoid cells. No obvious difference was recognized in these tissues between the IBM-BMT and IV-BMT groups (data not shown).

The process of regeneration in the tibia is illustrated in Figure 5A. The insertion of a needle into the tibia destroyed the bone marrow tissue to some extent, resulting in a small tissue defect with hemorrhage. At the site of destruction, the regeneration of the marrow architecture was observed around day 4 after BMT. At that time, the accumulation of hemopoietic cells was evident at the site of marrow regeneration in the IBM-BMT group. In the IV-BMT group, however, the numbers of BM stromal cells and hemopoietic cells were lower than in the IBM-BMT group, and the regeneration of marrow tissue was poor (Fig. 5B). The cellularity of the tibiae on day 4 was compared in the BMC-injected tibiae in the IBM-BMT group and the saline-injected tibiae in the IV-BMT group. Ten different areas were randomly selected in each tibia to analyze the cellularity. In total, three BMC-injected tibiae and three saline-injected tibiae were examined. The BMC-injected tibiae contained a significantly (p < .005) higher number of hemopoietic cells (92.1 ± 22.5/10 different areas of the tibia) than the saline-injected tibia (18.6 ± 3.1/10 different areas of the tibia). The hemopoietic cells were composed of myeloid cells, lymphoid cells, erythroid cells, and megakaryocytes. Moreover, when the number of stromal cells was compared in both tibiae, a significantly (p < .05) higher number of stromal cells (32.6 ± 4.4/10 different areas of the tibia) was found in the BMC-injected tibia than in the saline-injected tibia (18.7 ± 3.6/10 different areas of the tibia). The BM cavities were filled with the hemopoietic cells in all tibiae and femora by day 9 after BMT; at that time, no difference was observed between the groups or between any bones (data not shown).

View larger version (67K): [in this window] [in a new window]   Figure 5. Kinetics of histology. (A): Kinetics of histology of BM- or saline-injected tibiae in IBM-BMT and IV-BMT groups, respectively. Magnification, x4. The insertion of a needle into the tibia destroyed the BM tissue, resulting in a small tissue defect with hemorrhage. At the site of destruction, regeneration of the marrow architecture was observed at approximately day 4 after BMT in the IBM-BMT group. (B): The tibiae of IBM-BMT and IV-BMT on day 4. Magnification, x10 and x20. The regeneration of marrow tissue consists of absorption of dead bone fragments, osteoneogenesis, angiogenesis, and stromal proliferation. The accumulation of hemopoietic cells was evident at the site of marrow regeneration in the IBM-BMT group. In the IV-BMT group, however, a poor regeneration of marrow tissue was observed. Abbreviations: BM, bone marrow; BMC, bone marrow cell; BMT, bone marrow transplantation; IBM, intrabone-marrow; IV, intravenous.

To examine in detail whether the BMC-injected tibiae in the IBM-BMT group contained a higher number of donor-type stromal cells than the saline-injected tibiae in the IV-BMT group, we compared the number of donor-type stromal cells in the BMC-injected tibiae with that in the saline-injected tibiae on days 2, 4, and 6 after BMT. BM-adherent cells were obtained by culture of BMCs collected from the tibiae, and the adherent cells were double-stained with anti-H-2D^d (donor-type) mAb plus anti-CD45 mAb. H-2D^d-positive and CD45-negative cells were considered donor-type stromal cells. On day 2, the number of donor-type stromal cells per the tibia was 1.54 ± 0.43 x 10⁵ in the BMC-injected tibia and 0.98 ± 0.22 x 10⁵ in the saline-injected tibia (p < .05); 1.19 ± 0.36 x 10⁵ and 0.52 ± 0.28 x 10⁵ (p < .05) on day 4; 1.21 ± 0.93 x 10⁵ and 0.16 ± 0.07 x 10⁵ (p < .05) on day 6, respectively. This result clearly suggests that the BM-injected tibia contains a significantly higher number of donor-type stromal cells.

Homing of Stromal Cells
We have shown previously that when donor BM stromal cells are injected directly into the bone cavity, they proliferate at the site of injection [8, 11]. We therefore examined whether chemotactic factors capable of promoting the migration of BM stromal/mesenchymal stem cells are produced from BMCs soon after irradiation and also whether the factors are detected in the sera of the mice. For the migration assay, the FMS/PA6-P cell line was used, because we have shown previously that this cell line has characteristics of stromal cells and mesenchymal stem cells [13]. As shown in Figure 6A, no significant chemotactic activity was observed in the sera collected between 4 hours and 4 days after irradiation, whereas BM tissue extracts showed a significant chemotactic activity as early as 4 hours, reaching a maximum at 16 hours. In all BMT experiments performed in our laboratory, the donor BMCs are injected into the recipient mice around 16 hours after irradiation [15]. Therefore, it is conceivable that the BM stromal cells injected directly into the bones by IBM-BMT can settle at the site of injection and that only a small number of stromal cells leak and migrate anywhere. From these findings, it is unlikely that BM stromal cells enter into systemic circulation by IV-BMT and home into the recipient bone cavity.

View larger version (31K): [in this window] [in a new window]   Figure 6. Migration assay of stromal cells. (A): Stimulatory effect of BM tissue extracts obtained from irradiated mice on the migration of stromal cell line. Sera and bone marrow cells (BMCs) were collected from 9.5 Gy-irradiated C57BL/6 mice at the indicated time points after irradiation. The BMCs were cultured for 24 hours, and the culture supernatants were used as BM tissue extracts. FMS/PA6-P cells were assayed for migration toward the sera and BM tissue extracts. *, Number of adherent cell colonies per sample well/number of adherent cell colonies per control (sera and BM tissue extracts from normal mice) well. (B): Inhibition of the migration of stromal cell line toward HGF and BM tissue extracts by anti-HGF antibody. FMS/PA6-P cells were assayed for migration toward HGF and BM tissue extracts. In some wells, anti-HGF antibody was added in the lower well containing HGF or BM tissue extracts. *, Number of adherent cell colonies per sample well/number of adherent cell colonies per control (medium alone or BM tissue extracts from normal mice) well. Abbreviations: Ab, antibody; BM, bone marrow; HGF, hepatocyte growth factor; hr, hours; N.S., not significant; rhHGF, recombinant human hepatocyte growth factor.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
We have shown previously in [C57BL/6MRL/lpr] chimeric mice that the percentages of the donor-derived hemopoietic cells (even c-kit⁺/Lin^– cells) in the BM, spleen, and liver of IBM-BMT–treated mice are significantly higher than those in IV-BMT–treated mice within 2 weeks of BMT [8]. The number of donor-derived hemopoietic progenitor cells concomitantly increased, and donor-derived stromal cells were clearly detected in the cultured bone pieces from MRL/lpr mice treated with IBM-BMT. All the recipients thus treated survived for more than 1 year (>60 weeks after birth) and remained free from autoimmune diseases. In contrast, all the IV-BMT–treated mice died within 26 weeks after BMT. In addition, we have observed in the present study [B10AB10BR] that the numbers of donor-derived BMCs in the IBM-BMT–treated recipients are significantly higher than those in the IV-BMT–treated recipients as early as 3 days after BMT, although there is no significant difference in the spleen.

To clarify whether orthotopic BMC transplantation (IBM-BMT) is superior to heterotopic transplantation (IV-BMT) and why IBM-BMT has advantages over IV-BMT in which conventional IV-BMT does not result in successful engraftment, we analyzed the kinetics of hemopoietic regeneration after IBM-BMT from cytological and histological points of view.

As illustrated in Figures 1, 2B, and 3, significantly higher numbers of donor-derived cells were observed at days 2 through 6 after BMT at the site of the direct injection of BMCs. The data in Figure 1C showed that approximately 98% of injected cells had leaked away immediately after IBM-BMT. The leaked BMCs seem to have entered into other bones and proliferated there, because the regeneration in the contralateral bones and other bones was similar. We and other researchers have recently found that when BMCs are injected by the IV route, most donor BMCs (particularly pluripotent HSCs and stromal cells [mesenchymal stem cells]) are trapped in the lung after injection, and less than 5% enter the bone marrow cavity [1, 4, 18, 19]. These findings suggest that the seeding efficiency of the leaked BMCs into other bones is very low; therefore, the direct injection of BMCs into the bone cavity is an effective transplantation method.

The rate of increment in the donor-derived Lin^–/c-kit⁺ cells was very large, whereas that in the donor-derived Lin^–/CD34⁺ cells was very small (Fig. 3). Such differences probably reflect the differences in the dynamics of the populations (i.e., Lin^–/CD34⁺, Lin^–/c-kit⁺, and day 8 CFU-S). From these findings, it is assumed that BM progenitor cells quickly change their characteristics according to the stage of differentiation and the phase in the cell cycle. Another possibility is that the population of donor-derived Lin^–/CD34⁺ cells contains stromal cells and/or vascular endothelial cells. In the normal physiological state, the percentage of stromal cells and/or vascular cells in the BM is very low compared with that of hemopoietic cells, whereas the percentage of stromal cells and/or vascular cells increases in the BM during the early period after BMT.

After the injection of BMCs or saline into the left tibiae, the microarchitecture of the BM was destroyed, followed by the regeneration of hemopoietic marrow tissue (Fig. 5A). The process of marrow regeneration in this study was comparable with that in the heterotopic bone graft, where four sequential responses were identified in a spatially and temporally restricted manner: necrotic, clearing, stromal-proliferating, and hemopoiesis-recovering responses, as we reported previously [20]. In the present study, the stromal proliferating process observed on days 2 through 4 (Fig. 5) in the IBM-BMT group was higher than that in the IV-BMT group. Indeed, a more donor-type stromal cells were found in the BMC-injected sites than in the saline-injected sites on days 2 through 6. During this period, the accumulation of hemopoietic cells was higher in the tibiae at the site of the BMC injection than at the site of saline injection. This accumulation of hemopoietic cells explains the higher numbers of donor-derived cells in total and of Lin^–/c-kit⁺ cells at the site of injection of BMCs. Taken together, it is obvious that IBM-BMT causes a unique process in the early period after BMT, which is not observed in sham-operated tibiae in the IV-BMT group. Considering that the timing is crucial in ontogeny [21, 22] and that regeneration recapitulates ontogeny [20, 23–25], this early difference might explain why IBM-BMT is advantageous.

Askenasy demonstrated isolated limb perfusion with a BMC suspension to establish localized engraftment of donor BMCs [26]. He showed a similar pattern of donor cell distribution in the IBM-BMT and IV-BMT groups and hypothesized that IBM-BMT is, in fact, a systemic route of injection. This notion is compatible with our findings. Considering the capacity of the marrow space in the murine femoral or tibial bone, spillover must occur on the infusion of a large amount of fluid (0.2 ml in his case). This is why the intraosseous route has been used as an alternative for the systemic transfusion of fluids [27, 28].

The fate of the BMCs delivered into a systemic circulation via IV injection [1–3] or via spillover from IBM injection is still a matter of controversy. When nonadherent low-density BMCs were intravenously injected into the lethally irradiated syngeneic recipient mice, substantial proportions of injected BMCs were detected in the lung, liver, and marrow at 4 hours after injection and in the liver and marrow at 24 hours after injection [2]. Likewise, many of the IV-injected BM stromal cells were trapped in the lung (Y. Adachi, personal communication).

Many factors affect the engraftment of donor BMCs. There is a possibility that the direct delivery of donor BMCs by IBM-BMT facilitates the engraftment of early and late progenitor cells and, as a result, a higher number of hemopoietic cells were detected at the sites of the BMC injection. It is well known, however, that BM stromal cells are very important for hemopoiesis, especially for supporting HSCs. Although BM stromal cells are not as sensitive to irradiation as hemopoietic progenitor cells, the BM stromal cells are somewhat damaged after a lethal dose of irradiation. Moreover, we have demonstrated previously that major histocompatibility complex restriction exists between HSCs and BM stromal cells [29, 30]. Therefore, if BM stromal cells contained in nonirradiated donor BMCs can home effectively into the recipient bone cavity, the hemopoiesis of donor HSCs would be greatly advanced. In this sense, the direct injection by IBM-BMT of BMCs containing stromal cells would provide a feasible method. Indeed, we have observed that BMCs obtained from irradiated mice contain chemotactic factors capable of promoting the migration of stromal/mesenchymal cell line (FMS/PA6-P) (Fig. 6A), although sera from the irradiated mice are not prominent. BM stromal cells are known to produce HGF, and HGF is known to play an important role in hemopoiesis [31, 32]. Indeed, culture supernatants of BM stromal cells contain HGF at a concentration of 1–3 ng/ml without stimulation [32]. Therefore, it is conceivable that the BM tissue extracts obtained from irradiated mice contain HGF at a higher-than-normal concentration. The data in Figure 6B indicate that HGF contributes to the chemotactic activity of BM tissue extract, because the activity was inhibited by the addition of anti-HGF antibody. However, the activity was not completely abrogated by the antibody, indicating that chemotactic substances other than HGF are also contained in the BM tissue extracts.

We have recently carried out serial BMT to compare the potency of engraftment by IBM-BMT and IV-BMT. The data using [B6Ly5.1B6Ly5.2] chimeric mice indicate that IBM-BMT can accelerate the engraftment and proliferation of donor cells and, more importantly, can maintain donor DC subset up to at least the tertiary recipients [33]. We are now carrying out another serial BMT using [BALB/cC3H] chimeric mice. We have so far obtained the following results. The hematolymphoid system of tertiary recipients treated with IBM-BMT recovered earlier than that of tertiary recipients treated with IV-BMT, and, importantly, the progenitor cells of donor origin were well maintained in the tertiary recipients after serial IBM-BMT but not after serial IV-BMT (M. Omae, M. Inaba, Y. Sakaguchi, M. Tsuda, J. Fukui, H. Iwai, T. Yamashita, S. Ikehara, manuscript in preparation).

In summary, the difference between BMC-injected sites and other bone sites is detectable in the early phase (<1 week after BMT) of regeneration at the site of BM injection. During this period, the accumulation of hemopoietic cells is observed in concert with the regeneration of marrow microarchitecture. Precise characterization of the regenerating cell populations—both hemopoietic and environmental—at this particular time is necessary.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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We thank Y. Tokuyama, K. Hayashi, and A. Kitajima for their expert technical assistance. We also thank Hilary Eastwick-Field and K. Ando for their help in the preparation of the manuscript. This work was supported by a grant from "Haiteku Research Center" of the Ministry of Education, a grant from the "Millennium" program of the Ministry of Education, Culture, Sports, Science and Technology, a grant from the "Science Frontier" 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, and Health and Labor Sciences research grants (Research on Human Genome, Tissue Engineering Food Biotechnology), and also 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).

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