K. D. Patrene

Bone marrow stem cells develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of hepatocytes, biliary cells, or oval cells during liver regeneration. Cross-sex or cross-strain bone marrow and whole liver transplantation were used to trace the origin of the repopulating liver cells. Transplanted rats were treated with 2-acetylaminofluorene, to block hepatocyte proliferation, and then hepatic injury, to induce oval cell proliferation. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated hepatic cells were shown to be donor-derived. Thus, a stem cell associated with the bone marrow has epithelial cell lineage capability.

A recombinant retroviral vector (MFG-GC) was used to study the efficiency of transduction of the human gene encoding glucocerebrosidase (GC; D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45), in mouse hematopoietic stem cells and expression in their progeny. Transfer of the GC gene to CFU-S (spleen cell colony-forming units) in primary and secondary recipients was virtually 100%. In mice 4-7 months after transplantation, highly efficient transfer of the human gene to bone marrow cells capable of long-term reconstitution was confirmed by detection of one or two copies per mouse genome in hematopoietic tissues and in cultures of pure macrophages. Expression of the human gene exceeded endogenous activity by several fold in primary and secondary CFU-S, tissues from long-term reconstituted mice, and explanted macrophages cultures. These studies are evidence of the feasibility of efficient transfer of the GC gene to hematopoietic stem cells and expression in their progeny for many months after reconstitution. The results of this study strengthen the rationale for gene therapy as a treatment for Gaucher disease.

Evidence in experimental models suggests that many autoimmune diseases can be prevented by transplantation of bone marrow from disease-resistant donors. For potential clinical application, it would be important to avoid the morbidity and mortality associated with lethal conditioning and achieve mixed chimerism using less than complete recipient ablation. We report here for the first time that stable chimerism achieved in NOD mice using a sublethal radiation-based conditioning approach is sufficient to prevent beta-cell destruction and abrogate insulitis in prediabetic NOD mice. The percentage of NOD mouse recipients (8 wk of age) that engrafted with donor bone marrow correlated with the dose of irradiation and number of bone marrow cells transplanted. Engraftment of B10.BR bone marrow occurred in > or = 94% of animals receiving > or = 750 cGy of total body irradiation before bone marrow transplantation and > or = 30 x 10(6) bone marrow cells, while reproducible engraftment did not occur at radiation doses of less than 700 cGy and cellular doses of less than 30 x 10(6) bone marrow cells. All chimeric animals remained free of diabetes (n = 38) for 10 mo following bone marrow transplantation. Moreover, in all animals examined, no insulitis was present from 12 to 36 wk following reconstitution. In striking contrast, 61% (22 of 36) of NOD recipients that were conditioned but did not receive bone marrow developed acute diabetes by 12 mo. Insulitis was present in all remaining animals. These results suggest that allogeneic chimerism achieved using a sublethal conditioning approach can prevent the onset of diabetes and even reverse preexisting insulitis in NOD mice.

For over 40 years, the association between hemopoietic chimerism and donor-specific tolerance for allografts has been recognized. However, toxicity associated with lethal conditioning has prevented the clinical application of bone marrow (BM) chimerism to induce tolerance. We previously demonstrated that engraftment could be achieved with less than total recipient myeloablation (700 cGy) and that the incidence of engraftment correlated with the dose of total body irradiation (TBI). Administration of cyclophosphamide (CyP) on Day +2 reduced the minimum TBI dose sufficient to permit engraftment to 500 cGy. In the current study, addition of antilymphocyte globulin (ALG) to the TBI/CyP-based conditioning approach reduced the radiation required for engraftment to < or = 300 cGy. B10 (H-2b) mice conditioned with ALG on day -3, 300 cGy of TBI with transplantation of B10.BR (H-2k) or BALB/c (H-2d) BM on day 0, and CyP on day +2 exhibited evidence of donor chimerism (49.6 +/- 3.7% and 38.2 +/- 2.4%, respectively) in 97% of recipients. ALG eliminated CD4+ and CD8+ cells and decreased NK1.1+ cells in the peripheral circulation at the time of transplantation. Moreover, T and NK cells in the host BM were significantly decreased compared with cells of recipients conditioned with TBI alone. CyP delayed repopulation of host thymocytes, providing time for the establishment of donor chimerism before production of mature T cells. Chimeric animals exhibited stable multilineage chimerism and donor-specific tolerance to skin grafts and in in vitro assays. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.

Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic (A+B-->A) bone marrow results in multilineage mixed allogeneic chimerism, donor-specific transplantation tolerance, superior immunocompetence and resistance to graft-vs-host disease. However, the morbidity and mortality associated with lethal irradiation would be a major limitation to the clinical application of chimerism to induce tolerance for solid organ grafts or treat other nonmalignant hematologic diseases. We report here that durable multilineage mixed allogeneic chimerism and donor-specific transplantation tolerance for skin and primarily vascularized allografts can be achieved across multiple histocompatibility barriers using a nonmyeloablative radiation-based approach. The percentage of B10 mouse recipients that engrafted directly correlated with the degree of disparity between donor and recipient and the dose of total body irradiation administered. Although the occurrence of engraftment following conditioning with doses of total body irradiation of > or = 600 cGy was similar for animals receiving bone marrow disparate at MHC or MHC, minor and hematopoietic (Hh-1) loci (67% vs 78%), the level of donor chimerism was significantly less when multiple histocompatibility barriers were present (94.6 +/- 3.8% vs 37.5 +/- 12.5%). Treatment of the recipient with cyclophosphamide 2 days following allogeneic bone marrow transplantation reduced the dose of radiation sufficient for reliable engraftment to only 500 cGy of total body irradiation, regardless of MHC and Hh-1 disparity. Donor chimerism was stable and present in all lineages, with production of lymphoid (T and B cell), NK, and myeloid (erythrocyte, platelet, granulocyte, and macrophage) cells. Mixed chimeras exhibited donor-specific tolerance in vitro, as assessed by mixed lymphocyte culture (MLR) and cytotoxicity (CML) assays, and in vivo to skin and primarily vascularized cardiac allografts. The observation that engraftment and tolerance can be achieved across multiple histocompatibility barriers using nonmyeloablative recipient conditioning may allow allogeneic bone marrow transplantation to be applied to nonmalignant disease states in which lethal conditioning cannot be justified, including the induction of donor-specific tolerance for solid organ transplantation and the treatment of hemoglobinopathies and enzyme deficiency states.