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The long-term maintenance of hematopoietic stem cells (HSCs) is assessed by serial bone marrow transplantation (BMT), in which HSCs are injected intravenously. Recently, we have found that intra-bone marrow (IBM)-BMT can efficiently reconstitute the hematopoietic system with cells of donor origin, in contrast to conventional intravenous (i.v.) BMT. In the present study, we have compared the long-term maintenance of HSCs using multiple rounds of serial i.v.-BMT and IBM-BMT. The frequencies of donor-derived progenitor cells (Lin(-)/c-kit(+) cells) and more primitive progenitors (Lin(-)/c-kit(+)/CD34(+)/Sca-1(+) cells) were higher in the tertiary recipients by serial IBM-BMT than in those that had received bone marrow cells by serial i.v.-BMT. Furthermore, neither donor-derived progenitor cells nor mature hematolymphoid cells were detected in approximately 25% of the tertiary recipients after serial i.v.-BMT, indicating that progenitor cells can be efficiently maintained by IBM-BMT but not by i.v.-BMT. Finally, we confirmed that the recipients treated with the primary IBM-BMT (without carrying out serial BMT) showed a significantly higher survival rate than those treated with i.v.-BMT. These findings clearly show that IBM-BMT efficiently promotes the longterm maintenance of donor-derived hematopoiesis.

Background: Donor-specific central tolerance in cardiac allograft can be induced by hematopoietic chimerism via conventional intravenous bone marrow transplantation (IV-BMT). However, there are problems with IV-BMT, such as the risk of graft failure and of the toxicity from conditioning regimens., Methods: A new method for heart transplantation is presented. This method consists of administration of fludarabine phosphate (50 mg/kg) and fractionated low-dose irradiation (3.5 Gyx2 or 4.0 Gyx2), followed by intrabone marrow injection of whole bone marrow cells (IBM-BMT) plus heterotopic heart transplantation., Results: Cardiac allografts with IBM-BMT were accepted and survived long-term (>10 months) showing neither acute rejection nor chronic rejection including cardiac allograft vasculopathy by such conditioning regimens. In contrast, cardiac allografts with conventional IV-BMT were rejected within 1 month after the treatment with irradiation of 3.5 Gyx2 or within 3 months after the treatment with irradiation of 4.0 Gyx2. Macrochimerism (>70%) was favorably established and stably maintained by IBM-BMT but not IV-BMT. Low levels of transient mixed chimerism (<7%) were induced by IV-BMT with fludarabine plus 4.0 Gyx2, but the chimerism was lost within 1 month after the treatment., Conclusions: These findings indicate that IBM-BMT is a feasible strategy for the induction of persistent donor-specific tolerance, enables the use of reduced radiation doses as conditioning regimens, and obviates the need for immunosuppressants.

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra-bone marrow (IBM) injection of BMCs (termed IBM-bone marrow transplantation) has also been confirmed using 30 monkeys. Disclosure of potential conflicts of interest is found at the end of this article.

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 [IBM-BMT]). More than 1 month after IBM-BMT, hematolymphoid cells were completely reconstituted by donor-derived cells, and bone marrow stromal cells that could differentiate into osteocytes were also found to be of donor origin. In addition, the recipient C57BL/6 mouse showed the features of osteoporosis in the trabecular bone. Decreases in BMD and increases in urinary Dpd were also observed. When the message levels of cytokines (interleukin [IL]-11, IL-6, receptor activator of NF-kappaB ligand [RANKL], osteoprotegerin, macrophage-colony-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." Disclosure of potential conflicts of interest is found at the end of this article.

We have recently found that intra-bone marrow-bone marrow transplantation (IBM-BMT) can be used to prevent graft-versus-host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM-BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5 Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM-BMT plus i.v. injection of allogeneic T cells (intravenous DLI [IV-DLI]). GvHD in recipients treated with allogeneic IBM-BMT plus IBM-DLI was far milder than in those treated with allogeneic IBM-BMT plus IV-DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4+CD25+ and CD4+Foxp3+ cells was significantly higher in recipients treated with IBM-BMT plus IBM-DLI than in those treated with IBM-BMT plus IV-DLI. Donor-derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM-BMT plus IBM-DLI, whereas Th1 cells were dominant in recipients treated with IBMBMT plus IV-DLI. Furthermore, the production of transforming growth factor-β and hepatocyte growth factor from bone marrow stromal cells was enhanced after IBM-DLI. Thus, IBM-BMT plus IBM-DLI seem to preferentially induce Tregs and Th2, resulting in the prevention of GvHD. [ABSTRACT FROM AUTHOR]