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RAPID COMMUNICATION |
Center for Light Microscope, Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA; Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA; Present address: Frankel Laboratory of Bone Marrow Transplantation, Center of Pediatric Hematology Oncology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel
Key Words. Bone marrow transplantation • Intra-bone marrow • Isolated limb perfusion • Hematopoietic chimerism • Skin grafts • Tolerance
Correspondence:
Nadir Askenasy, Ph.D., Frankel Laboratory of Bone Marrow Transplantation, Center of Pediatric Hematology Oncology, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petach Tikva, 49202, Israel. Telephone: 9-723-925-3669; Fax: 9-723-925-3042; e-mail: anadir{at}012.net.il
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Extending these considerations, it was hypothesized that localized BMT could improve the efficiency of engraftment in nonmyeloablated recipients. There is mounting evidence that a megadose of donor cells improves engraftment across antigenic barriers, promotes engraftment of purified hemopoietic stem cells (HSC), and reduces the requirement for conditioning [12–15]. A "megadose" effect may be generated either by infusion of a large number of cells or inoculation of a limited hematopoietic space. It was recently demonstrated that IB-BMT reversed autoimmunity in MRL/lpr mice [7]. In this study, IB- and isolated limb perfusion (IL)-BMT were assessed as strategies for induction of localized BMT, and their tolerogenic efficiency was tested by skin grafting. The results indicated that IB inoculation is equivalent to i.v. injection of cells, while engraftment is localized after IL-BMT. Both routes of administration induced tolerance to skin grafts in nonmyeloablated recipients.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intra-Bone Marrow Transplantation
The knee was exposed by a sharp skin incision, flexed, and a 24G needle was inserted into distal femoral epiphysis above the patella (Fig. 1
). Donor BMC were injected into the femur using a miniperistaltic pump (P720; Instech; Plymouth Meeting, PA; http://www.instechlabs.com) via a double outlet system. Cells were infused through one outlet at a rate of sim;20-50 µl/minutes, and the other outflow was connected to a threshold pressure transducer and a PowerLab monitoring system (ADInstruments; Grand Junction, CO; http://www.adinstruments.com). When intraluminal pressure raised above a threshold value (8 or 20 mmHg), the medium was diverted into a drainage reservoir.
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). Surgery was performed under magnification using a Leica GZ6 surgical stereoscope (Leica; Northvale, NJ; http://www.leica.com). The KH perfusion medium was aerated with a mixture of CO2/O2 5/95% to give a final pH of 7.3, and was warmed to 37°C in a double-walled water-jacketed container [16]. The femoral artery was perfused in three stages at a rate of 0.2 ml/minute using a miniperistaltic pump: the first 5 minutes with KH, then with donor cells in KH (0.5 x 106-108 cells/ml), and the last 10 minutes with KH. The low infusion volume aimed to minimize extravasation and accumulation of cells in the soft tissues of the limb. Outflow from the femoral vein was collected into a container and was occluded after perfusion, diverting blood drainage from the limb to the deep veins. The femoral artery was restored either by a side-to-side anastomosis, ligation to a short segment of the 24G catheter, or repair of the site of cannule insertion. The limb was gradually reperfused by intermittent release of the arterial clamp.
Isolation of BMC
BMC were harvested from femurs and tibia crushed in Hank' balanced salt solution (HBSS; GIBCO Laboratories; Grand Island, NY; http://www.invitrogen.com). BMC were suspended using an 18G needle, filtered with a 30 µm sterile nylon mesh, collected by centrifugation (400 g, 10 minutes, 4°C) and resuspended in HBSS containing 2% fetal calf serum (FCS). Erythrocytes were lysed by incubation with ammonium chloride for 4 minutes at room temperature. Nucleated cells (whole BMC) were counted after being washed twice with excess medium.
T-cell depletion was performed by incubation for 30 minutes at 4°C with rat-anti-mouse CD4 and CD8 monoclonal antibodies (mAb; Pharmingen; San Diego, CA; http://www.pharmingen.com). Excess mAb were removed by washing twice with PBS containing 2% FCS. Then, cells were gently mixed for 20 minutes at 4°C with goat-anti-rat immunoglobulin G conjugated to M-450 magnetic beads at a ratio of four beads per cell (Dynal Inc.; Lake Success, NY; http://www.dynal.no). T cells rosetted with beads were precipitated by exposure to a magnetic field, and supernatant containing nonlymphocytes was collected. Depletion of T cells was assessed by flow cytometry (Coulter Elite; Miami, FL; http://www.coulter.com) using fluorescein isothiocyanate (FITC)-labeled anti-
ßT-cell receptor mAb.
PKH Staining
2 x 107 cells were suspended in 1 ml of Diluent C, and freshly prepared PKH67 membrane linker was added to a final concentration of 2 µM (provided by Dr. K. Muirhead; SciGro Co.; Malvern, PA; http://www.maconsultants.com/scigro). Samples were incubated at room temperature for 5 minutes with gentle mixing. Staining was terminated by addition of 4 volumes HBSS containing 10% FCS, cells were collected by centrifugation (400 g, 10 minutes, 4°C) and washed twice with HBSS. The average recovery of the procedure was 90%, with a viability of 95% as determined with the trypan blue exclusion test.
Kinetics of BMC Systemic Scattering
Blood samples were collected into heparinized serum vials in 200 µl HBSS, washed twice, and collected by centrifugation (400 g, 10 minutes, 4°C). Femurs were harvested from mice euthanized by CO2 asphyxiation under anesthesia, and BMC were collected from bones crushed in HBSS. After lysis of erythrocytes, the fraction of PKH+ cells was determined by flow cytometry using the blast cell gate. When the number of PKH+ cells was low, samples were screened with a fluorescence microscope (Axiophot; C. Zeiss; Thornwood, NY; http://www.zeiss.com).
Characterization of Donor Chimerism
Blood samples were collected into heparinized serum vials in 200 µl HBSS and were incubated with fluorochrome-labeled mAb: anti-H2b-PE mAb for B10 donors, anti-H2k-FITC and H2d-FITC mAb for B10.BR and BALB/c recipients, respectively. Blood cells were layered over 1.5 ml lymphocyte separation media (1.087 g/ml; CedarLane; Hornby, Ontario, Canada; http://www.cedarlanelabs.com). After centrifugation (1,000 g, 20 minutes, 4°C), low-density cells were collected, washed twice with HBSS, and fixed with 0.5% paraformaldehyde. The percentage of phycoerythrin (PE)+-donor (H2b) peripheral blood lymphocytes (PBL) was determined by flow cytometry on the lymphocyte gate.
Skin Grafting
Full-thickness tail skin from BMC-matched and third party donors was grafted in the inter-scapular region of anesthetized chimeras [17]. Grafts were inspected on a daily basis for signs of rejection. Disappearance of the epidermis was considered as complete rejection.
Statistical Analysis
Data are presented as means ± standard deviation for each experimental protocol. Within the groups, reproducibility was evaluated by linear regression of duplicate measurements. Differences between the experimental protocols were estimated with a post hoc Scheffe t-test at a 5% level of significance.
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RESULTS |
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Three Approaches to BMT: Myeloablated Recipients
None of the myeloablated B10.BR mice (busulfan 145 µg/g) injected with medium (without BMC) survived for 30 days. Injection of 5 x 107 syngeneic whole BMC (B10.BR
B10.BR) i.v., IB, and IL rescued all myeloablated recipients. Survival of myeloablated B10.BR recipients after i.v. injection of allogeneic BMC (B10B10.BR) was 100% and 90% after IL and IB injection (n = 10). At 4 weeks, the chimeras had 96% ± 4%, 91% ± 5%, and 78% ± 4% donor lymphocytes in the i.v., IB, and IL protocols, respectively. The data demonstrated the capacity of IL- and IB-BMT to reconstitute hematopoiesis in myeloablated recipients.
The Fate of BMC: IB Injection Versus IL Perfusion
The early systemic distribution of BMC labeled with PKH membrane linkers was assessed in peripheral blood and femurs of the recipients. Preliminary studies showed that PKH dyes did not affect, qualitatively or quantitatively, cellular homing and seeding in recipient BM. Figure 2A compares the dissipation of PKH+ cells in peripheral blood after i.v., IB, and IL transplantation of syngeneic BMC in myeloablated B10.BR recipients. The fraction of PKH+ blast cells was not significantly different between the i.v. and IB protocols (n = 5). In both cases, PKH+ cells almost disappeared from the blood within 2 hours after transplantation. Even distribution of transplanted cells could result either from their systemic scattering during IB injection, or early mobilization of cells from the injected femur and secondary systemic dispersion. Lowering the injection pressure to 8 mmHg (n = 3) did not change the profile of PKH+ cells in peripheral blood. In contrast, very few PKH+ cells were detected in peripheral blood of mice injected IL (p < 0.05 versus i.v.- and IB-BMT), demonstrating limited systemic dissipation.
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). First, there were no significant differences between the number of PKH+ cells in the injected femur (IB) and the contralateral naïve femur (Fig. 3). Second, the fraction of PKH+ BMC in the IB protocol was similar to the contents of both femurs after i.v.-BMT. Third, the profile did not change when the IB injection pressure was lowered to 8 mmHg. These data indicated that, in our experimental conditions, IB and i.v. infusions were equivalent in terms of systemic BMC dissipation.
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). Detailed examination of the contralateral femur by fluorescence microscopy revealed 12 ± 3 (range 8-37) PKH+ cells. Considering the low levels of PKH+ BMC in the blood after IL-BMT, it was evident that systemic scattering was markedly reduced in the IL protocol. In a separate group of mice, femurs were harvested 48 hours after IL-BMT (n = 4), and sim;70 PKH+ BMC were identified by fluorescence microscopy in the noninjected femur.
IL-BMT in Nonmyeloablated Recipients
An attractive application of localized BMT is induction of hemopoietic chimerism to achieve tolerance to donor-matched organs. To evaluate the characteristics of IL-BMT, three variables were assessed: nonablative recipient preconditioning, size and composition of donor inoculum, and timing of BMC and skin grafting. Survival of BALB/c recipients conditioned with doses of 35-145 µg/g busulfan 36 hours before i.v. infusion of medium (without cells) is presented in Figure 4A. Injection of 107 whole BMC from B10 donors rescued mice conditioned with all doses of busulfan (n = 5). Overall, higher doses of busulfan increased the levels of donor PBL at 4 weeks post-transplantation (Fig. 4B).
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BALB/c) into recipients conditioned with 70 µg/g busulfan increased the levels of donor chimerism (Fig. 4C). A sharper increase in donor PBL occurred in the range of 106-107 whole BMC injected i.v., and 1-5 x 106 BMC injected IL (n = 5). The lower levels of chimerism, and the smaller numbers of BMC that increased the yield of engraftment, may reflect a hemopoietic space limitation during IL infusion.
The less efficient engraftment of T-cell-depleted BMC (TCD-BMC) may be improved by infusion of large numbers of cells [12,14]. To assess potential advantages of the megadose effect, BALB/c recipients conditioned with 70 µg/g busulfan were injected with 107 whole and TCD-BMC (n = 10). While the fraction of donor PBL decreased by 35% when TCD-BMC were injected i.v. (p < 0.001 versus whole BMC), there was a nonsignificant decrease of 10% in mice perfused IL (Fig. 4D). Overall, IL infusion of TCD-BMC resulted in superior levels of donor PBL compared with i.v. injection (p < 0.05). The 30-day survival rates were 80% and 90% after i.v. and IL transplantation of TCD-BMT, respectively.
Acceptance of Secondary Skin Grafts
The tolerogenic effect of IL-BMT was assessed by secondary transplantation of skin grafts 3 weeks after BMT (B10BALB/c). All recipients conditioned with 70 µg/g busulfan injected i.v., IB, and IL with 107 whole BMC accepted donor-matched skin grafts for periods exceeding 16 weeks (n = 5). In a similar manner, skin grafts were accepted by all viable chimeras transplanted with TCD-BMC either i.v. or IL (n = 5). In contrast, third party skin allografts (B10.BR) were acutely rejected in these experimental groups (n = 3-4).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, IL perfusion led to localized engraftment in the limb and progressive secondary systemic seeding of transplanted cells. IL transplantation shared some of the characteristics of i.v.- and IB-BMT. Infusion of syngeneic and allogeneic BMC rescued myeloablated recipients, and the levels of donor PBL chimerism varied with conditioning and the size of donor inoculum. At 4 weeks, the IL and i.v. protocols induced comparable levels of donor PBL chimerism, which were sufficient for acceptance of skin allografts by nonmyeloablated recipients. Creation of localized BMT was evident from the remarkable increase in femur cellularity in the perfused limb, and the extremely low incidence of PKH+ cells in peripheral blood and contralateral femurs. Considering that IL-BMT created localized engraftment and systemic distribution of few cells, it is likely that BMC were mobilized from the injected limb at a later time [18]. Secondary systemic redistribution was apparent in the levels of donor chimerism at 30 days (24%-76%) that exceeded by far the contribution of one femur and tibia (7%-9%) to the hemopoietic space in the mouse [19].
Infusion of a large number of cells into a small hematopoietic space creates a local "megadose effect" that may provide significant advantages for initial HSC engraftment [12–15]. It may be argued that if transplanted cells have to compete with host HSC for vacant niches for engraftment, most prominent in nonablated recipients, then there may be a limitation in number of BMC that can be effectively transplanted into a limb [11,13]. However, there was no apparent limitation in stromal niches for initial seeding when large numbers of BMC (2 x 108) were injected directly into the lumen of isolated femurs [20].
We found several advantages of IL, as a route of administration of donor cells. First, the results support the prediction that the efficiency of localized engraftment would increase at a smaller donor inoculum in the IL- versus i.v.-BMT [13–15]. Second, there was a significant improvement in efficiency of engraftment of TCD-BMC infused into the isolated limb compared with injection into a peripheral vein. Depletion of T cells decreased the incidence of GVHD, however, more conditioning was required for engraftment [21,22]. Infusion of a megadose of donor cells was shown to overcome the absence of T cells and antigenic barriers between donor cells and recipient BM stroma [7,12]. It will be interesting to further assess whether IB- and IL-BMT reduce the reactivity of T cells that leads to GVHD, as recently reported for IB marrow infusion [7]. Third, immunoreactivity against third party allografts was preserved, suggesting that acceptance of the skin grafts was mediated by hemopoietic chimerism [1–5], and was not a limited consequence of chemical conditioning. Finally, considering the relative safety of IL [23], IL-BMT may have significant clinical applications for induction of tolerance and treatment of nonmalignant disorders.
In this study, busulfan was chosen as a single conditioning agent for assessment of the localized BMT approach, because its cytoreductive effect is more pronounced than its immunosuppressive activity. The assumption was that localized BMT significantly reduces the need for systemic immunosuppression at the time of transplantation. Busulfan is a cytoreductive agent that selectively affects slow-cycling and quiescent BMC, predominantly primitive stem cells in G0 [24]. It is likely that the systemic preconditioning procedure used in this study promoted the rates of secondary systemic seeding of locally transplanted BMC [18,25].
In summary, this study reports a novel approach for localized BMT by IL. The data demonstrate that IL-BMT: A) resulted in initial localization of donor cells and secondary expansion to give high levels of donor chimerism; B) reconstituted hematopoiesis in myeloablated recipients; C) induced hemopoietic chimerism as efficiently as systemic BMT, and D) led to donor-specific tolerance to secondary skin grafts across major and minor antigenic barriers, while preserving immunoreactivity against third party alloantigens. This approach may have clinical applications for treatment of nonmalignant disorders and induction of tolerance without myeloablative conditioning.
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ACKNOWLEDGMENT |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A) in rats. Cell Transplant
1993;2:345–353.[Medline]This article has been cited by other articles:
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  Q. Li, H. Hisha, R. Yasumizu, T.-X. Fan, G.-X. Yang, Q. Li, Y.-Z. Cui, X.-L. Wang, C.-Y. Song, S. Okazaki, et al. Analyses of Very Early Hemopoietic Regeneration After Bone Marrow Transplantation: Comparison of Intravenous and Intrabone Marrow Routes Stem Cells, May 1, 2007; 25(5): 1186 - 1194. [Abstract] [Full Text] [PDF]
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 K. T. Chabner, G. B. Adams, J. Qiu, M. Moskowitz, E. S. Marsters, G. P. Topulos, and D. T. Scadden Direct vascular delivery of primitive hematopoietic cells to bone marrow improves localization but not engraftment Blood, June 15, 2004; 103(12): 4685 - 4686. [Full Text] [PDF]
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K. Nakamura, M. Inaba, K. Sugiura, T. Yoshimura, A-H. Kwon, Y. Kamiyama, and S. Ikehara Enhancement of Allogeneic Hematopoietic Stem Cell Engraftment and Prevention of GvHD by Intra-Bone Marrow Bone Marrow Transplantation Plus Donor Lymphocyte Infusion Stem Cells, March 1, 2004; 22(2): 125 - 134. [Abstract] [Full Text] [PDF]
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N. Askenasy, E. S. Yoleuk, H. Shirwan, Z. Wang, and D. L. Farkas Cardiac Allograft Acceptance after Localized Bone Marrow Transplantation by Isolated Limb Perfusion in Nonmyeloablated Recipients Stem Cells, March 1, 2003; 21(2): 200 - 207. [Abstract] [Full Text] [PDF]
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N. Askenasy and D. L. Farkas Optical Imaging of PKH-Labeled Hematopoietic Cells in Recipient Bone Marrow In Vivo Stem Cells, November 1, 2002; 20(6): 501 - 513. [Abstract] [Full Text] [PDF]
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N. Askenasy, T. Zorina, D. L. Farkas, and I. Shalit Transplanted Hematopoietic Cells Seed in Clusters in Recipient Bone Marrow In Vivo Stem Cells, July 1, 2002; 20(4): 301 - 310. [Abstract] [Full Text] [PDF]
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