Analyses of Very Early Hemopoietic Regeneration After Bone Marrow Transplantatio - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:55 |只看该作者 |倒序浏览 |打印

作者：Qing Lia,d, Hiroko Hishaa,b,c, Ryoji Yasumizua, Tian-Xue Fana, Guo-Xiang Yanga, Qiang Lia, Yun-Ze Cuia, Xiao-Li Wanga, Chang-Ye Songa, Satoshi Okazakia, Tomomi Mizokamia, Wen-Hao Cuia, Kequan Guoa, Ming Lia, Wei Fenga, Junko Katoua, Susumu Ikeharaa,b,c


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   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., X. a3 |4 k) j" w4 h5 S2 I% T; J

Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Intravenous-bone marrow transplantation Intrabone marrow-bone marrow transplantation Hemopoietic regeneration Chemotaxis4 O9 R# z( E+ b. |4 [2 e0 ~" m
   INTRODUCTION
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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 .8 ?1 p: s5 f% x3 u
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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 . Thus, it is conceivable that stromal cells provide an advantage for hemopoiesis when injected directly into bone cavity.* n* ?7 G- y+ ~; S$ Y

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.% p6 R+ e/ O: A- M# S9 ^

MATERIALS AND METHODS

Mice

Seven week-old female B10.A (H-2a), B10.BR (H-2k), BALB/c (H-2d), and B57BL/6 (H-2b) 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
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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¨C18 hours before BMT. In some experiments, mice were irradiated (9.5 Gy) to get their sera and BMCs at various time points after irradiation.2 q* w. ~( c- e# S! Q" A' N  s. R

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.
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Bone Marrow Transplantation4 U2 @* e0 @$ n
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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.+ A3 T- W. D3 ~$ V0 f; M6 W5 ?, f

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% s$ W2 l% X1 H* O6 n
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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-2Dd monoclonal antibody (mAb) for B10.A mice or phycoerythrin (PE)-conjugated anti-H-2Kd 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-2Dk mAb for B10.BR mice or FITC-conjugated anti-H-2Kb 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-2Kb 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.4 ^  p) X6 L; W0 Z! q: ^5 p
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Colony-Forming Unit in Spleen
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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  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% |* |9 C+ ~' f' ^& ]
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BMCs were collected from the tibiae of the recipient mice of  on days 2, 4, and 6 after BMT (5¨C7 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-2Dd mAb (donor-type) plus anti-CD45 antibody (BD Pharmingen). H-2Dd-positive and CD45-negative cells were considered donor-type stromal cells.9 Z) v' S! ?3 \5 \

Collection of Sera and BM Tissue Extracts from the Irradiated Mice
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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.+ B) P1 q3 c) l; @; @

Migration Assay of Stromal Cells4 i, A7 B- c* v/ |
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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 : 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¨C20 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.; L$ @; V: c1 t4 N# g+ r

In our preliminary experiments, sera or BM tissue extracts were placed in the lower chamber at concentrations of 5¨C50%, and their chemotactic activity was examined for FMS/PA6-P cells. Maximum migration was observed at the concentrations of 10%¨C20% 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.  E% i9 R: N6 _
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Statistical Analyses. A0 s# Z9 a- T, G1 a

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.
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RESULTS( v/ c+ J% P5 A

Kinetics of Donor-Derived Hemopoiesis

In the first set of experiments, we compared the kinetics of regeneration of bone marrow cells in the  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-2Dd mAb (donor-type) and anti-H-2Dk mAb (recipient-type), and analyzed by FACScan.% P) B$ Q, \9 {8 a# |# U7 u

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 : t; B' g% k$ G  u. ]  d! Y. c
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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.4 U( M  z' J# u0 r! N
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In Figure 1, the data were plotted only for the tibiae and without error bars to simplify the graph. The humeri and femora showed almost the same kinetics as the contralateral tibiae of the IBM-BMT group or both tibiae in the IV-BMT group (data not shown). In the kinetics of the hemopoietic regeneration in the spleens, no significant difference was observed between the IBM-BMT and IV-BMT groups (data not shown). In the second set of fully allogeneic bone marrow chimeras  chimeras (data not shown).

Kinetics of "Lymphocyte," "Blast," and "Granulocyte" Window Cells5 h; T/ Y3 l! o! A1 A

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) , where most of the cells in each window are B lymphocytes, immature myeloid progenitor cells, or granulocytes, respectively.  m# C& c) P1 J

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.. f1 c6 h; t5 k8 e1 d1 b
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Donor-derived cells in the tibia of mice that had received IBM- and IV-BMT were analyzed according to these windows on the SSC/FSC dot plot profile. The donor-derived cells in all three windows at the site of BMC injection in the IBM-BMT group (Fig. 2B) started to increase earlier than at the site of saline injection in the IV-BMT group (Fig. 2C). All other BM sites that had not been injected with donor BMCs showed a pattern similar to that in Figure 2C. The differences between the IBM-BMT and IV-BMT groups were not obvious on day 9 and thereafter.6 T1 k. R4 \1 D" A
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Kinetics of Lin¨C/CD34  and Lin¨C/c-kit  Cells+ f7 Y) a$ W1 q: }; q8 x

To trace the hematopoietic precursor cells, donor-derived BMCs were further divided into Lin¨C/CD34  (Fig. 3A) and Lin¨C/c-kit  cells (Fig. 3B) in  chimeras. As shown in Figure 4A, Lin¨C/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/c-kit  cells in the BM-injected tibiae were statistically significant (p

Figure 3. Kinetics of the regeneration of donor-derived Lin¨CCD34  (A) and Lin¨Cc-kit  (B) cells in tibia after IBM-BMT or IV-BMT. The number of donor-derived Lin¨Cc-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
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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
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Kinetics of Day 8-CFU-S. T3 s4 C  h% H( l$ Y0 w
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Because the total BMC number was limited during the early period after BMT, only the day 8-CFU-S assay was performed in the combination of . We confirmed in our preliminary experiments that only donor-derived BMCs (not endogenous CFU-S) contribute to the formation of colonies in this assay (unpublished observations).

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
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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$ m# r4 l/ C0 Y6 |
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After severe depletion of lymphocytes as early as 1 day after BMT, the thymus and lymph nodes slowly recovered cellularity by days 9¨C14. 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).3 Q$ \( H7 d( t0 v  a8 V! c
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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 & s( a9 M8 }1 N1 M

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.3 M, X3 A2 k, Z4 `

We also examined the histological kinetics of other bones (humeri, femora, ribs, vertebral bodies, and ilium). Each site was analyzed at 3 and 8 hours, and 1, 2, 4, 6, 9, and 14 days after BMT, and the experiments were repeated more than 10 times. These bone sites showed similar kinetics to the contralateral tibiae in the IBM-BMT group or tibiae on both sides (left and right) in the IV-BMT group.

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-2Dd (donor-type) mAb plus anti-CD45 mAb. H-2Dd-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 ¡À 0.43 x 105 in the BMC-injected tibia and 0.98 ¡À 0.22 x 105 in the saline-injected tibia (p 0 C  t& k$ a9 T5 w9 Z1 U

Homing of Stromal Cells
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We have shown previously that when donor BM stromal cells are injected directly into the bone cavity, they proliferate at the site of injection . 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.
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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.
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Some reports indicate that human and mouse mesenchymal stem cells express c-met (a receptor of HGF) and have a characteristic of migrating toward HGF in a Transwell migration assay . Therefore, we investigated the possibility that HGF is contained in the BM tissue extracts and takes part in the chemotactic activity (Fig. 6B). When rhHGF was added to the lower chamber at various concentrations, the migration of FMS/PA6-P was observed in a dose-dependent manner. The activity was completely abrogated by the addition of anti-human HGF antibody. The BM tissue extracts collected 16 hours after irradiation also showed chemotactic activity. When anti-human HGF antibody was added to the BM tissue extracts, the activity was partially, but not completely, inhibited. This suggests that the BM tissue extracts contain active substances other than HGF.

DISCUSSION

We have shown previously in  that the numbers of donor-derived BMCs in the IBM-BMT¨Ctreated recipients are significantly higher than those in the IV-BMT¨Ctreated recipients as early as 3 days after BMT, although there is no significant difference in the spleen.
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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.5 H( V+ S9 t2 N8 |: K

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 . 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.
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The rate of increment in the donor-derived Lin¨C/c-kit  cells was very large, whereas that in the donor-derived Lin¨C/CD34  cells was very small (Fig. 3). Such differences probably reflect the differences in the dynamics of the populations (i.e., Lin¨C/CD34 , Lin¨C/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¨C/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 , this early difference might explain why IBM-BMT is advantageous.' o3 Y* O* V  h  u& C

Askenasy demonstrated isolated limb perfusion with a BMC suspension to establish localized engraftment of donor BMCs .
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The fate of the BMCs delivered into a systemic circulation via IV injection . Likewise, many of the IV-injected BM stromal cells were trapped in the lung (Y. Adachi, personal communication).9 }4 }6 o0 P% v" V! z& g

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 . 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., S# r/ H' o$ Q( l
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We have recently carried out serial BMT to compare the potency of engraftment by IBM-BMT and IV-BMT. The data using  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).0 S& l: Z) U& x$ m! W
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In summary, the difference between BMC-injected sites and other bone sites is detectable in the early phase (0 u# y' M: ?, [: `6 ?/ `
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST- t/ g+ Y" z, }+ q" I0 G
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The authors indicate no potential conflicts of interest., Y7 P; ]. q% e) ~4 y

ACKNOWLEDGMENTS( X1 x* h' R; \: s  t$ W
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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).9 h2 f* O/ C5 ?
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