We have previously demonstrated that stromal cells can support the proliferation and differentiation of hematopoietic cells in vitro and in vivo and that a major histocompatibility complex restriction exists between hematopoietic stem cells and stromal cells. We have also found that intra–bone marrow (IBM) injection of allogeneic bone marrow cells (BMCs) leads to more rapid reconstitution of hematopoietic cells than intravenous injection. In the present study, we examine the effect of simultaneous injection of stromal cells and BMCs into the same bone marrow on the recovery of donor hematopoietic cells and demonstrate that simultaneous IBM injection of BMCs plus stromal cells is more effective in reconstituting recipients with donor hematopoietic cells than intravenous injection of BMCs plus stromal cells or IBM injection of BMCs alone.
It has been reported that bone marrow stromal cells (BMSCs) play a crucial role in hematopoiesis and that BMSCs support hematopoietic cells by cognate interaction and production of humoral factors [1–5]. Even in vitro, Dexter culture using BMSCs can maintain hematopoiesis for longer than 6 months . Moreover, several cell lines derived from the bone marrow have been reported to support hematopoiesis [7–9]. MC3T3-G2/PA6 (PA6), a mouse bone marrow stromal cell line established from newborn mouse calvaria, can also support hematopoiesis in vitro [10, 11]. We have previously reported that the transplantation of bone marrow cells (BMCs) plus BMSCs successfully ameliorates autoimmune diseases in MRL/Mp-lpr/lpr (MRL/lpr) mice, which possess radioresistant abnormal hemopoietic stem cells . In the experiment, we injected allogeneic BMCs into a vein and transplanted a flushed bone under the renal capsule as a source of BMSCs. These could then migrate from the transplanted bone to the host bone marrow, thereby supporting hematopoiesis of the donor's BMCs. We have also found that a major histocompatibility complex (MHC) restriction exists between bone marrow hematopoietic stem cells and BMSCs [13–15]. Recently we have found that the intra–bone marrow (IBM) injection of allogeneic BMCs ameliorates intractable autoimmune diseases in MRL/lpr mice .
In this study, we demonstrate that the IBM injection of BMCs plus BMSCs promotes the hematopoiesis of donor BMCs, resulting in prolongation of survival.
Materials and Methods
C57BL/6 (B6, H-2b) mice and C3H/HeN (C3H, H-2k) mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). B6 mice carrying the eGFP transgene (eGFP+ B6 mice) were kindly donated by Dr. H. Okano (Osaka University, Osaka, Japan) . All of the mice were maintained in a pathogenfree environment.
The recipient mice (B6 and C3H) were exposed to 9.5 Gy of gamma irradiation from a 137Cs source (Gammacell 40 Exactor; Nordion International Inc., Kanata, Ontario, Canada) 1 day before transplantation. Injection of 1 × 105 BMCs from eGFP+ B6 mice or 1 × 105 PA6 into the bone marrow (IBM–bone marrow transplantation [BMT]) or the vein (IV-BMT) of recipient mice was performed as described previously . The following groups were prepared: only radiation for C3H mice, IBM-BMT of PA6 to C3H mice, IBM-BMT of eGFP+ B6 BMCs to C3H mice, IV-BMT of eGFP+ B6 BMCs plus PA6 to C3H mice, IBM-BMT of eGFP+ B6 BMCs plus PA6 to C3H mice, IBM-BMT of eGFP+ B6 BMCs to B6 mice, IBM-BMT of saline to C3H mice, IBM-BMT of latex beads to C3H mice, IBM-BMT of eGFP+ B6 BMCs plus latex beads to C3H mice, IBM-BMT of eGFP+ B6 BMCs plus fixed PA6 to C3H mice, IBM-BMT of B6 BMSCs to C3H mice, and IBM-BMT of eGFP+ B6 BMCs plus B6 BMSCs to C3H mice. To prepare fixed PA6, PA6 was fixed with 4% paraformaldehyde for 30 minutes on ice. After washing three times with phosphate-buffered saline, the cells were used as fixed PA6. Latex beads (3 μm) were purchased from Sigma-Aldrich, Inc. (St. Louis).
Colony-Forming Unit of Spleen Assays
The recipient mice were euthanized on day 12 after transplantation, and their spleens were removed, weighed, and fixed in Bouin's solution. Visible surface colonies were counted 1 day after fixation.
Analyses for Numbers of White Blood Cells, Red Blood Cells, and Platelets in Peripheral Blood
The peripheral blood (PB) was analyzed using an SF-3000 autoanalyzer for the PB (Sysmex, Kobe, Japan).
Analyses of Chimerism
Twelve days after transplantation, the peripheral WBCs were phenotyped for recipient/donor cells by flow cytometry. In brief, the PB of the recipient mice was collected, and the WBCs were isolated by discontinuous density-gradient centrifugation using Lymphocyte-mammal (Cedarlane, Ontario, Canada). The WBCs were incubated with phycoerythrin (PE)–conjugated anti-CD45 monoclonal antibody (BD Biosciences, San Jose, CA). The stained cells were analyzed using a FACScan (Becton, Dickinson, Mountain View, CA). CD45+/eGFP+ cells in CD45+ cells were considered as donor-derived WBCs.
Analyses of BMCs
BMCs were obtained from BMC-injected tibias in IBM-BMT groups and one randomly selected tibia in each of the other groups. First, numbers of total BMCs were counted. Next, BMCs were stained with TC-labeled anti-CD45 antibody (Ab; Caltag, Burlingame, CA) plus PE-labeled anti-CD11b Ab (BD Biosciences), TC-labeled anti-CD45 Ab plus PE-labeled anti-B220 Ab (BD Biosciences), or TC-labeled anti-CD45 Ab plus PE-labeled anti-Ter119 Ab (BD Biosciences). Stained cells were analyzed using FACStar. Percentages of eGFP+ cells in CD45+ cells, eGFP+ cells in CD11b+ cells, eGFP+ cells in B220+ cells, or eGFP+ cells in Ter119+ cells in the bone marrow were examined. Total cell numbers of each type of surface marker–positive cell were calculated using the total cell number of the tibia and the percentage of each fraction.
Culture of BMSCs
We cultured freshly isolated BMCs of B6 mice and expanded BMSCs according to the methods of Fan et al. .
Significances were evaluated using the Student's t-test, except for survival rate. Significances of survival rates were evaluated using the log-rank test. Probability values of less than .05 were considered to be statistically significant.
Increase in Colony-Forming Unit of Spleen Counts on Day 12 of Mice That Received IBM-BMT of BMCs Plus Stromal Cells
To examine whether MHC-matched stromal cells accelerate hematopoiesis in vivo, we injected eGFP+ B6 BMCs and PA6 into a bone marrow cavity or a tail vein of lethally irradiated C3H mice, followed by determination of colony-forming unit of spleen (CFU-S) on day 12. As shown in Figure 1, simultaneous IBM injection of BMCs from eGFP+ B6 mice plus PA6 promoted a significant increase of day-12 CFU-S compared with IBM-BMT of only BMCs or IV-BMT of BMCs plus PA6. However, day-12 CFU-S counts in the IBM-BMT of BMCs plus PA6 group still did not attain the counts of the syngeneic IBM-BMT (IBM-BMT of eGFP+ B6 mice into B6 mice) group. We obtained similar data when we used BMCs from B6 mice instead of BMCs from eGFP+ B6 mice.
Early Recovery of Donor Hemopoietic Cells in Mice That Received IBM-BMT of BMCs Plus Stromal Cells
We counted the number of WBCs, RBCs, and platelets in the PB of the recipients to examine the recovery of hematopoiesis 12 days after transplantation. As shown in Figures 2A–2C, the BMCs plus PA6 group treated with IBM-BMT showed good recovery of hematopoiesis compared with the groups treated with IV-BMT of BMCs plus PA6 or IBM-BMT of only BMCs. Although there were no significant differences between these groups because of their large standard deviations, we repeatedly noted good recovery in the group treated with IBM-BMT of BMCs plus PA6.
Next, to examine whether transplanted BMCs differentiate into mature hemopoietic cells, we determined the percentage of eGFP+ nuclear cells in the PB of the host mice. As shown in Figure 2D, a significantly high percentage of eGFP+ WBCs was found in the PB of mice that received IBM-BMT of eGFP+ BMCs plus PA6 compared with mice that received IBM-BMT of only BMCs or IV-BMT of BMCs plus PA6. The mean percentage of eGFP+ WBCs in the PB of mice in the IBM-BMT of BMCs plus PA6 group was approximately 50%, whereas the PB of mice in the group that received IBM-BMT of only BMCs or IV-BMT of BMCs plus PA6 showed only approximately 20% of donor cells. There were also significant differences between the groups treated with IBM-BMT of BMCs plus PA6 and IV-BMT of BMCs plus PA6 or IBM-BMT of BMCs. These results suggest that IBM-BMT of BMCs plus MHC-matched stromal cells is effective in promoting the hematopoiesis of donor BMCs.
Next, we also analyzed the recovery of donor hematopoietic cells in the bone marrow. We examined the BMC-injected bone (tibia) of the IBM-BMT groups and randomly selected tibias in other groups. As shown in Figure 3, total number of bone marrow cells, percentage of eGFP+ cells, number of eGFP+ cells, number of CD11b+ cells, number of B220+ cells, and number of Ter119+ cells increased to a greater extent in the group treated with IBM-BMT of BMCs plus PA6 compared with other groups. In fact, there was no significant difference between some groups, but we observed a clear tendency toward better hematopoietic recovery in the group treated with IBM-BMT of BMCs plus PA6.
Prolonged Survival in Mice That Received IBM-BMT of BMCs Plus Stromal Cells
We next examined whether IBM-BMT of BMCs plus MHC-matched stromal cells had the ability to prolong the survival of transplanted host mice. As shown in Figure 4, the survival rate of mice that received IBM-BMT of BMCs plus PA6 was approximately 60%, even on day 60, by which time mice in all groups other than syngeneic IBM-BMT had already died. These results suggest that rapidly developed donor hematopoietic cells enabled the mice to survive in the IBM-BMT of BMCs plus PA6 group and that sufficient hematopoiesis did not occur in other groups, except for syngeneic BMT, because the dead mice had shown no symptoms and no pathological findings of graft versus host disease. Even in the IBM-BMT of BMCs group, in which we previously observed more rapid hematopoiesis than in the IV-BMT BMCs group, the mice died rapidly, probably because of the very small number of BMCs transplanted .
Simultaneously Injecting Cultured BMSCs Plus BMCs into Same Bone Accelerates Hematopoiesis
To apply this method clinically, we used BMSCs instead of PA6 in our system. Because there are very few BMSCs in freshly isolated BMCs, we cultured BMSCs to enrich and to augment the number of BMSCs . Next, we injected cultured BMSCs plus BMCs into the tibias to examine whether cultured BMSCs can accelerate hematopoiesis. As shown in Figure 5, cultured BMSCs plus BMCs showed higher CFU-S counts and heavier spleen weight, suggesting significantly enhanced hematopoiesis. We also prepared several kinds of control to exclude the possibility of nonspecific physical stimulation. Saline was used as a control for volume expansion, and latex beads were used for the control of volume expansion and physical stimulation. Neither injection of saline nor latex beads augmented the CFU-S counts or spleen weight. IBM-BMT of latex beads plus BMCs or IBM-BMT of fixed PA6 plus BMCs resulted in a smaller number of CSF-S than IBM-BMT of only BMCs, suggesting that latex beads and fixed PA6 disturbed hematopoiesis.
There are numerous reports discussing the role of BMSCs in hematopoiesis [1–15]. Interestingly, the BMSCs support hematopoietic cells by means of not only humoral factors but also direct interaction. Thus, cell-to-cell interaction between BMSCs and hematopoietic cells is crucial for hematopoiesis. Our newly established method for BMT is to inject the BMCs into the bone marrow of the host, which accelerates hematopoiesis, resulting in the rapid recovery of hematopoiesis by the transplanted BMCs . This method enables us to inject hematopoietic cells (including hematopoietic stem cells) plus stromal cells (including mesenchymal stem cells) directly into the bone marrow, thereby placing these cells in an ideal position for hematopoiesis. It has been shown that CFU-S counts on day 12 reflect the number of pluripotent hemopoietic stem cells [14, 19]. Therefore, we compared day-12 CFU-S counts using the various BMT methods to analyze their effects on hematopoiesis. When we injected BMCs plus PA6 into the bone marrow, day-12 CFU-S counts increased significantly compared with IV-BMT of BMCs plus PA6 or IBM-BMT of only BMCs. These results suggest that injected stromal cells accelerate hematopoiesis in the transplanted bone marrow even in vivo.
It has been reported that PA6 cells support hematopoiesis through cell-to-cell interaction, but not by the conditioned medium of PA6 cells [20, 21]. In fact, PA6 cells have been reported to express stem cell antigen-1, macrophage colony-stimulating factor (M-CSF), and stem cell factor (SCF) on their surface . In our experiment, only live PA6 cells (not fixed PA6 cells) accelerated hematopoiesis. These results suggest that PA6 cells support hematopoiesis through surface molecules that PA6 cells continuously produce. On the other hand, it has also been reported that BMSCs express CD44, vascular cellular adhesion molecule-1, MECA10, and stromal cell–derived factor-1 and produce interleukin (IL)-1β, IL-7, M-CSF, SCF, insulin-like growth factor-1, and leukocyte inhibitory factor . We showed that not only the PA6 stromal cell line, but also cultured BMSCs have an effect on hematopoiesis in vivo. Therefore, to apply this method clinically, we must use live BMSCs obtained from the same donor as the BMCs.
The key point of this method is the simultaneous injection of BMCs plus stromal cells into the same bone marrow, where they are able to come into contact with each other. It has been reported that intravenously injected cells are trapped in the lungs and liver [24–26]. Indeed, when we injected PA6 into the vein, most were trapped in the lungs (data not shown).
Therefore, this method is clinically applicable, because precultured BMSCs plus freshly harvested BMCs should be directly injected into the bone marrow cavity of recipients (IBM-BMT). This method is expected to accelerate hematopoietic recovery, thereby reducing the incidence of rejection and graft failure.
We thank Ms. Murakami-Shinkawa, Ms. Tokuyama, and Ms. Miura for their expert technical assistance and Mr. Eastwick-Field and Ms. Ando for the preparation of this manuscript. This study was supported by grants from Haiteku Research Center of the Ministry of Education, grant-in-aid for scientific research (B) 11470062, grant-in-aid for scientific research (Hoga) 16659107, grants-in-aid for scientific research on priority area (A) 10181225 and (A) 11162221, a grant from Millennium of Ministry of Education, Culture, Sports, Science and Technology, and a grant from the Scientific Frontier program of the Ministry of Education, Culture, Sports, Science and Technology. The Department of Transplantation for Regeneration Therapy, Kansai Medical University, was sponsored by Otsuka Pharmaceutical Company, Ltd.
Yuming Zhang and Yasushi Adachi contributed equally to this work.