Photochemical treatment of donor lymphocytes inhibited their ability to facilitate donor engraftment or increase donor chimerism after nonmyeloablative conditioning or establishment of mixed chimerism.

Donor T-cells can provide a graft-versus-leukemia effect and help to promote donor engraftment after allogeneic BMT; however, these benefits can be outweighed by the ability of the cells to induce life-threatening GVHD. Photochemical treatment (PCT) of T-cells with S-59 psoralen and long-wavelength UV-A light can inhibit their proliferative capacity and significantly decrease their ability to induce acute GVHD after allogeneic BMT. PCT donor T-cells have been shown to facilitate donor engraftment in a myeloablative BMT model. In this study, we examined whether donor T-cells subjected to PCT ex vivo could retain the ability to facilitate engraftment or increase donor chimerism after nonmyeloablative BMT or after establishment of mixed hematopoietic chimerism. In a transplantation model in which mice were conditioned for BMT with sublethal (600 cGy) TBI, an infusion of PCT donor T-cells was unable to facilitate engraftment of donor BM. A BMT model was used in which a mixture of allogeneic and syngeneic marrow cells was infused into lethally irradiated recipients for establishment of mixed hematopoietic chimerism. The goal was to determine whether PCT donor splenocytes could increase levels of donor chimerism. Recipients of splenocytes treated with UV-A light only (no S-59 psoralen) and given at the time of BMT or in a donor lymphocyte infusion (DLI) had significantly higher levels of donor chimerism than did recipients of BM only. Although PCT donor splenocytes given at the time of BMT modestly increased donor chimerism, PCT donor splenocytes given in a DLI did not increase donor chimerism. A nonmyeloablative BMT model was employed for determining whether DLI given relatively late after BMT could increase donor chimerism. Recipient mice were conditioned for BMT with a combination of low-dose TBI (50 or 100 cGy) and anti-CD154 (anti-CD40L) monoclonal antibody for achievement of low levels of mixed chimerism. When control mixed chimeras were given a DLI 71 days after BMT, donor chimerism was significantly increased. In contrast, PCT of the donor cells eliminated the ability of the cells to increase donor chimerism after infusion. Together results from these 3 distinct BMT models indicate that PCT of donor T-cells significantly inhibited the ability of the cells to facilitate donor engraftment after nonmyeloablative BMT or to increase donor chimerism in mixed hematopoietic chimeras when the cells were administered in a DLI.     

The contribution of cytotoxic and noncytotoxic function by donor T-cells that support engraftment after allogeneic bone marrow transplantation.

The present studies were designed for investigation of the requirements for cytotoxic function in donor T-cells transplanted to support engraftment after infusion of allogeneic bone marrow. The experiments examined the capacity of donor CD8 T-cells lacking Fas ligand and/or perforin function to facilitate donor B6 congenic (B6-Ly5.1) BM engraftment across major histocompatibility complex class I/II barriers after transplantation. T-cell-depleted BM cells from B6-Ly5.1 donors were transplanted into sublethally irradiated (5.5 Gy) BALB/c recipients together with different lymphocyte populations from wild-type B6 (B6-wt) donors or donors lacking functional cytotoxic pathways. Early presence of lineage-committed donor progenitor cells was assessed by the presence of day 5 splenic colony-forming units-granulocyte-macrophage (CFU-GM). Recipients of BMT without donor T-cells did not demonstrate significant CFU-GM activity 5 days post-BMT. Lineage-committed progenitor cells in recipient spleens could be supported by addition to the BM of wild-type (B6-wt) and cytotoxically single- (perforin, B6-pko or FasL, B6-gld) or double-deficient (B6-cdd) CD8 T-cells. However, B220+-enriched B-cells could not support the presence of day 5 donor CFU-GM. For further assessment of the capacity of cytotoxically impaired T-cells to participate in the engraftment process, the ability of these and normal CD8 cells to support the homing of donor cells to the BM was examined after infusion of carboxyfluorescein diacetete succinimidyl ester-labeled progenitors. In a syngeneic model lacking resistance, cytotoxically impaired donor T-cells supported increased numbers of progenitor cells in the marrow equivalent to the support provided by wild-type donor T-cells. Examination of peripheral chimerism indicated that during the first month after B6-->BALB/c BMT, donor chimerism was detected in BMT recipients receiving unfractionated T-cells or CD8+ T-cells from B6-wt donors, and chimerism was maintained at least 80 days after BMT. In contrast, B6-cdd unfractionated or CD8+ T-cells failed to maintain long-term B6 donor chimerism in the host. Experiments with highly enriched populations of positively selected CD8+ T-cells from B6-pko, B6-gld, or B6-cdd donors demonstrated that although each of these T-cell populations could promote the initial presence of donor CFU-GM early post-BMT, B6-pko and B6-cdd CD8+ T-cell populations were not able to support long-term peripheral chimerism. These results demonstrate that donor T-cells lacking major cytotoxic effector pathways have functions that support initial donor progenitor cell presence in the host hematopoietic compartment after BMT. They also demonstrate that support of long-term donor BM engraftment requires CD8+ T-cells with intact cytotoxic, that is, perforin, function. Finally, syngeneic B6-->B6 BMT suggests activation of CD8+ T-cells posttransplantation apparently is required to support enhanced progenitor cell activity. This study provides new findings concerning the role of cytotoxic function in the process of facilitating allogeneic donor BM engraftment.     

Analyses of very early hemopoietic regeneration after bone marrow transplantation: comparison of intravenous and intrabone marrow routes

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. Disclosure of potential conflicts of interest is found at the end of this article.     

Intra-bone marrow injection of donor bone marrow cells suspended in collagen gel retains injected cells in bone marrow, resulting in rapid hemopoietic recovery in mice

We have recently developed an innovative bone marrow transplantation (BMT) method, intra-bone marrow (IBM)-BMT, in which donor bone marrow cells (BMCs) are injected directly into the recipient bone marrow (BM), resulting in the rapid recovery of donor hemopoiesis and permitting a reduction in radiation doses as a pretreatment for BMT. However, even with this IBM injection, some of the injected BMCs were found to enter into circulation. Therefore, we attempted to modify the method to allow the efficient retention of injected BMCs in the donor BM. The BMCs of enhanced green fluorescent protein transgenic mice (C57BL/6 background) were suspended in collagen gel (CG) or phosphate-buffered saline (PBS), and these cells were then injected into the BM of irradiated C57BL/6 mice. The numbers of retained donor cells in the injected BM, the day 12 colony-forming units of spleen (CFU-S) counts, and the reconstitution of donor cells after IBM-BMT were compared between the CG and PBS groups. The number of transplanted cells detected in the injected BM in the CG group was significantly higher than that in the PBS group. We next carried out CFU-S assays. The spleens of mice in the CG group showed heavier spleen weight and considerably higher CFU-S counts than in the PBS group. Excellent reconstitution of donor hemopoietic cells in the CG group was observed in the long term (>100 days). These results suggest that the IBM injection of BMCs suspended in CG is superior to the injection of BMCs suspended in PBS.     

The effect of the spleen on compartmental levels and distribution of donor progenitor cells after syngeneic and allogeneic bone marrow transplants

These studies investigate the involvement of the spleen in progenitor (PC) cell numbers and "cross-talk" with the marrow compartment following syngeneic or allogeneic bone marrow transplantation (BMT) in sham or fully splenectomized mice. Intact recipient B6 mice were lethally irradiated prior to transplant with T cell-depleted bone marrow (BM-TCD). The kinetics of PC reconstitution following i.v. transplant consistently revealed a dramatic increase in splenic colony-forming unit interleukin-3 (CFU IL-3) and CFU (high proliferative potential-(HPP) levels between days 5 and 12 post-BMT. Direct injection of TCD-BM into the recipient marrow cavity did not alter this pattern of reconstitution in the splenic compartment. In contrast to spleens from normal adult B6 mice containing 0.9% and 0.6% of the total combined splenic and marrow committed (CFU IL-3) and primitive (CFU-HPP) progenitors, respectively, spleens of syngeneic BMT recipients at day 12 contained a 10-fold increase (p < 0.001) over the progenitor levels in normal spleens. These splenic numbers decreased to normal, homeostatic levels by day 28 post-BMT. In contrast, the level of marrow CFU IL-3 progenitors continued to increase post-transplant, reaching near homeostatic levels by day 28 post-BMT. Interestingly, early seeding of 5- (and -6)carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled or green fluorescent protein (GFP) donor bone marrow cells (BMC) to the marrow compartment was not different in sham splenectomies or recipients splenectomized 14 days earlier. However, recipient splenectomy consistently resulted in significantly higher numbers of CFU IL-3 in the bone marrow during the first 2 weeks post-transplant compared to sham controls. These elevated levels exceeded the combined progenitor numbers of the splenic and marrow compartments of intact recipients. Notably, this increase in marrow progenitor activity in splenectomized recipients was observed after syngeneic as well as allogeneic BMT. Allogeneic transplants across major, or those limited to minor, histocompatibility antigen differences exhibited this increased marrow progenitor activity. Splenectomy performed 2 h post-transplant to assure "normal" marrow seeding also resulted in higher marrow progenitor activity. Thus, this "marrow response" to splenectomy is not induced by early "shunting" of infused BM cells to the marrow compartment. These results suggest that communication between the splenic and marrow compartments following syngeneic and allogeneic BMT exists during early hematopoietic reconstitution, one effect of which is to impact the compartmental distribution of donor progenitor cells. The role of the spleen on engraftment, chimerism, and tolerance in allogeneic BMT models are now under investigation.     

Enhancement of allogeneic hematopoietic stem cell engraftment and prevention of GVHD by intra-bone marrow bone marrow transplantation plus donor lymphocyte infusion

We examined the effect of intra-bone marrow (IBM)-bone marrow transplantation (BMT) in conjunction with donor lymphocyte infusion (DLI) on the engraftment of allogeneic bone marrow cells (BMCs) in mice. Recipients that had received 6 Gy of radiation completely rejected donor BMCs, even when IBM-BMT was carried out. However, when BMCs were IBM injected and donor peripheral blood mononuclear cells (PBMNCs) were simultaneously injected intravenously (DLI), donor cell engraftment was observed 7 days after BMT and complete donor chimerism continued thereafter. It is of interest that the cells of recipient origin did not recover, and that the hematolymphoid cells, including progenitor cells (Lin-/c-kit+ cells) in the recipients, were fully reconstituted with cells of donor origin. The cells in the PBMNCs responsible for the donor BMC engraftment were CD8+. Recipients that had received 6 Gy of radiation, IBM-BMT, and DLI showed only a slight loss of body weight, due to radiation side effects, and had no macroscopic or microscopic symptoms of graft-versus-host disease. These findings suggest that IBM-BMT in conjunction with DLI will be a valuable strategy for allogeneic BMT in humans.     

Multiple mixed chimeras: reconstitution of lethally irradiated mice with syngeneic plus allogeneic bone marrow from multiple strains

Reconstitution of lethally irradiated B10 mice with a mixture of 5 x 10(6) B10 plus 15 x 10(6) B10.D2 T-cell-depleted (TCD) bone marrow (BM) cells has previously been shown to produce stable, mixed chimeras which are specifically tolerant to donor skin grafts; the inclusion of TCD syngeneic marrow in the inoculum leads to improved immunocompetence in the resulting chimeras. In order to determine whether this method of transplant tolerance induction could be extended to multiple simultaneous allogeneic donors, we have investigated the engraftment capacity of combinations containing syngeneic and more than one allogeneic source of bone marrow. B10 mice were lethally irradiated and reconstituted with a mixture of (B10 + B10.D2 + B10.BR) or (B10 + B10.RIII + B10.BR) TCD BM. Analysis of each group of animals by flow microfluorometry provided evidence for stable multiple mixed chimerism in the majority of animals. All animals which exhibited such multiple chimerism were also tolerant of skin grafts from both allogeneic donors and promptly rejected fourth party skin grafts. An attempt to produce chimerism with TCD marrow from 5 allogeneic plus syngeneic BM cells was less successful. When animals were given non-TCD allogeneic BM from 2 allogeneic donors along with TCD syngeneic BM, they reconstituted as fully allogeneic chimeras in which one or the other allogeneic donor prevailed. These results indicate that (1) multiple allogeneic donor BM cells can engraft simultaneously in the mixed marrow model, but there may be a limit to the number of marrow strains which can repopulate a single animal; (2) multiple allogeneic engraftment confers transplantation tolerance to multiple donors; and (3) TCD is essential to permit multiple mixed chimerism to develop.     

The entry of the prothymocyte into the thymus after lethal irradiation and bone marrow transplantation. I. Seeding of bone marrow cells into the thymus

Bone marrow (BM) cells arrive in the thymus of lethally irradiated mice as early as three hours after bone marrow transplantation (BMT). They can be recognized by labeling of the injected cells with Hoechst 33342 (direct homing assay). In order to relate the immigrated BM cells to thymocyte precursor cells, direct homing and thymus repopulation experiments were compared. It was shown that homing of BM cells depends on the time between lethal irradiation and BMT, while it was previously shown that thymus repopulation does not. In addition, thymic immigrants were smaller than precursor cells committed to the T cell limeage (prothymocytes) and their progenitors. A cell population obtained from normal BM cells and enriched in stem cells (purified stem cells) was previously shown to repopulate the thymus similarly as BM cells from mice pretreated in vivo with 5-fluorouracil (FUBM). Both cell suspension showed a delayed thymus repopulation when compared to normal BM. This is indicative for a depletion of prothymocytes in these cell suspensions. In the direct homing assay, however, it was found that relatively many cells from FUBM seeded into the thymus, while purified stem cells did not. These results indicate that most if not all donor cells that are present in the thymus at three hours after BMT are not thymocyte precursor cells.     

Delayed Marrow Infusion in Mice Enhances Hematopoietic and Osteopoietic Engraftment by Facilitating Transient Expansion of the Osteoblastic Niche

Transplantation of bone marrow cells leads to engraftment of osteopoietic and hematopoietic progenitors. We sought to determine whether the recently described transient expansion of the host osteoblastic niche after marrow radioablation promotes engraftment of both osteopoietic and hematopoietic progenitor cells. Mice infused with marrow cells 24 hours after total body irradiation (TBI) demonstrated significantly greater osteopoietic and hematopoietic progenitor chimerism than did mice infused at 30 minutes or 6 hours. Irradiated mice with a lead shield over 1 hind limb showed greater hematopoietic chimerism in the irradiated limb than in the shielded limb at both the 6- and 24-hour intervals. By contrast, the osteopoietic chimerism was essentially equal in the 2 limbs at each of these intervals, although it significantly increased when cells were infused 24 hours compared with 6 hours after TBI. Similarly, the number of donor phenotypic long-term hematopoietic stem cells was equivalent in the irradiated and shielded limbs after each irradiation-to-infusion interval but was significantly increased at the 24-hour interval. Our findings indicate that a 24-hour delay in marrow cell infusion after TBI facilitates expansion of the endosteal osteoblastic niche, leading to enhanced osteopoietic and hematopoietic engraftment.     

Mesenchymal stem cell-organized bone marrow elements: an alternative hematopoietic progenitor resource

Bone marrow-derived mesenchymal stem cells (BMMSCs) are multipotent postnatal stem cells that have been used for the treatment of bone defects and graft-versus-host diseases in clinics. In this study, we found that subcutaneously transplanted human BMMSCs are capable of organizing hematopoietic progenitors of recipient origin. These hematopoietic cells expressed multiple lineages of hematopoietic cell associated markers and were able to rescue lethally irradiated mice, with successful engraftment in the recipient, suggesting a potential bone marrow (BM) resource for stem cell therapies. Furthermore, we found that platelet-derived growth factor (PDGF) promotes the formation of BMMSC-generated BM niches through upregulation of beta-catenin, implying that the PDGF pathway contributes to the formation of ectopic BM. These results indicate that the BMMSC-organized BM niche system represents a unique hematopoietic progenitor resource possessing potential clinical value.     

Crucial role of donor-derived stromal cells in successful treatment for intractable autoimmune diseases in mrl/lpr mice by bmt via portal vein

We have recently established a new bone marrow transplantation (BMT) method for the treatment of intractable autoimmune diseases in MRL/lpr mice; the method consists of fractionated irradiation (5.5 Gy x 2), followed by BMT of whole bone marrow cells (BMCs) from allogeneic C57BL/6 mice via the portal vein (abbreviated as 5.5 Gy x 2 + PV). In the present study, we investigate the mechanisms underlying the early engraftment of donor-derived cells in MRL/lpr mice by this method. In the mice treated with this method, the number of donor-derived cells possessing the mature lineage (Lin) markers rapidly increased in the BM, spleen, and liver; almost 100% were donor-derived cells by 14 days after the treatment. The number of donor-derived hemopoietic progenitor cells (defined as c-kit(+)/Lin(-) cells) increased in the BMCs, hepatic mononuclear cells, and especially spleen cells by 14 days after the treatment. Simultaneously, hemopoietic foci adjoining donor-derived stromal cells were observed in the liver when injected via the PV, but not via the peripheral vein (i.v.). When adherent cell-depleted BMCs were injected via the PV, recipients showed a marked reduction in the survival rate. However, when mice were transplanted with adherent cell-depleted BMCs with cultured stromal cells, all the recipients survived. These findings suggest that not only donor hematopoietic stem cells (HSCs) but also donor stromal cells administered via the PV were trapped in the liver, resulting in the early engraftment of donor HSCs in cooperation with donor-derived stromal cells. This new strategy to facilitate the early recovery of hemopoiesis would therefore be of great advantage in human application.     

Induction of tolerance in quadruple chimeric mice

Human cord blood (CB) contains hematopoietic stem cells and progenitors. Because the major limitation to a widespread use of CB for transplantation lies in its limited volume, it is necessary to combine the CB from several donors. In this study, we show that lethally irradiated mice can be reconstituted with the injection of a mixture of T cell-depleted bone marrow cells (BMCs; total, 3 x 10(6)) obtained from three fully allogeneic mouse strains in two different mouse combinations. A higher survival rate was obtained in the triple injection group than in mice injected with BMCs (1 x10(6)) obtained from a single mouse strain. In the mixed chimeric mice, three kinds of donor-type and recipient-type cells were detected in all the hematopoietic organs 1 month after bone marrow transplantation (BMT). Mixed-lymphocyte reaction showed that the tolerance to both recipient-type and donor-type major histocompatibility complex determinants was induced in the chimeric mice. In the peripheral blood (PB) of these mice, only one type of cells from the three different donor strains became dominant in most chimeric mice and reached a stable level about 4 months after BMT. Polymerase chain reaction analyses, however, revealed that the skins from all the donors were accepted even when no cells with their phenotypes could be detected in the PB. These results suggest that both hemato-lymphoid reconstitution and stable tolerance to not only the recipient strain but also all the donor strains can be achieved in chimeric mice, indicating the possibility of mixed CB transplantation in humans.     

Engraftment of donor-derived epithelial cells in multiple organs following bone marrow transplantation into newborn mice

Bone marrow-derived cells (BMDCs) can engraft as epithelial cells throughout the body, including in the lung, liver, and gastrointestinal (GI) tract following transplantation into lethally irradiated adult recipients. Except for rare disease models in which marrow-derived epithelial cells have a survival advantage over endogenous cells, the currently attained levels of epithelial engraftment of BMDCs are too low to be of therapeutic benefit. Here we tested whether the degree of bone marrow to epithelial engraftment would be higher if bone marrow transplantation (BMT) were performed on 1-day-old mice, when tissues are undergoing rapid growth and remodeling. BMT into newborn mice after multiple different regimens allowed for robust hematopoietic engraftment, as well as the development of rare donor-derived epithelial cells in the GI tract and lung but not in the liver. The highest epithelial engraftment (0.02%) was obtained in mice that received a preparative regimen of two doses of busulfan in utero. When BMDCs were transplanted into myelosuppressed newborn mice that lacked expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, the chloride channel that is not functional in patients with cystic fibrosis, the engrafted mice showed partial restoration of CFTR channel activity, suggesting that marrow-derived epithelial cells in the GI tract were functional. However, BMT into newborn mice, regardless of the myeloablative regimen used, did not increase the number of bone marrow-derived epithelial cells over that which occurs after BMT into lethally irradiated adult mice.     

Prevention of graft-versus-host disease by intrabone marrow injection of donor T cells: involvement of bone marrow stromal cells

We have developed a new and effective method for bone marrow transplantation (BMT): bone marrow cells (BMCs) are injected directly into the bone marrow (BM) cavity of recipient mice. The intrabone marrow injection of BMCs (IBM-BMT) greatly facilitates the engraftment of donor-derived cells, and IBM-BMT can attenuate graft-versus-host reaction (GVHR), in contrast to conventional intravenous BMT (i.v.-BMT). Here, we examine the mechanisms underlying the inhibitory effects of IBM-BMT on GVHR using animal models where GVHR is elicited. Recipient mice (C57BL/6) were irradiated and splenic T cells (as donor lymphocyte infusion: DLI) from major histocompatibility complex-disparate donors (BALB/c) were injected directly into the BM cavity (IBM-DLI) or injected intravenously (i.v.-DLI) along with IBM-BMT. The BM stromal cells (BMSCs) from these recipients were collected and related cytokines were examined. The recipient mice that had been treated with IBM-BMT + i.v.-DLI showed severe graft-versus-host disease (GVHD), in contrast to those treated with IBM-BMT + IBM-DLI. The suppressive activity of BMSCs in this GVHD model was determined. The cultured BMSCs from the recipients treated with IBM-BMT + IBM-DLI suppressed the proliferation of responder T cells remarkably when compared with those from the recipients of IBM-BMT + i.v.-DLI in mixed leucocyte reaction. Furthermore, the level of transforming growth factor-beta and hepatocyte growth factor in cultured BMSCs from IBM-BMT + IBM-DLI increased significantly when compared with those from the recipients of IBM-BMT + i.v.-DLI. Thus, the prevention of GVHD observed in the recipients of IBM-BMT + IBM-DLI was attributable to the increased production of immunosuppressive cytokines from BMSCs after interaction with host reactive T cells (in DLI).     

Degeneration of stroma reduces retention of homed cells in bone marrow of lethally irradiated mice

Cytotoxic drugs or irradiation are generally administered before bone marrow (BM) transplantation because of the idea that host bone marrow 'niches' become available to the donor cells for engraftment. How BM stromal cells respond to the radiation, which ultimately modulates grafting of donor cells, is poorly understood. In this study, we examined homing and marrow retention of PKH26+ donor cells in BM of age-matched C57BL/6J mice conditioned at different doses of irradiation. When we injected donor cells into mice that received 900 cGy, the percent homing was highest (15.8 +/- 1.5%) as compared to the lower doses of radiation. Despite the highest levels of homing of donor cells in these mice, about 70% (p < 0.005) homed cells were detached from the marrow within 72 h of transplantation. In contrast, a 2- to 2.5-fold (p < 0.03) multiplication of homed PKH-26+ Sca-1+ cells was observed in sublethally irradiated mice. While determining that CD45- CD106+ cells in BM of the mice received 900 cGy, we found that more than 80% of cells were depleted. It was also revealed from this investigation that grafted cells conferred partial protection to the endogenous myeloid colony-forming cells from radiation injury. Collectively, the present study implicates radiation-induced degeneration of stroma as a cause of poor retention of donor cells in BM of lethally irradiated mice. These results may have important clinical implications in designing conditioning regimens for BM transplantation.     

Reduction of marrow hematopoietic progenitor and stem cell content is not sufficient for enhanced syngeneic engraftment

The mechanisms regulating long-term engraftment of primitive stem cells are largely unknown. Most conditioning strategies use myeloablative agents for experimental or clinical hematopoietic stem cell transplantation. Host conditioning regimens, in part, have been designed on the assumption that transplanted cells home to specific marrow sites and if these sites are occupied by host stem cells, engraftment will not take place. However, there is now evidence that stable long-term syngeneic engraftment may occur in the absence of host marrow stem cell depletion. To further study the association of engraftment with stem cell depletion, we investigated whether the marked egress of hematopoietic progenitor and stem cells from the marrow into the peripheral blood in C57BL6 mice following a single dose of cyclophosphamide (day 1) and four days of G-CSF (days 3-6) afforded an increased opportunity for long-term syngeneic donor engraftment. During and after mobilization, glucose phosphate isomerase (GPI)-1(b) mice received 30 x 10(6) GPI-1(a) marrow cells without further myeloablation. The level of donor/recipient chimerism was assessed in cell lysates after six months. Increased long-term syngeneic donor engraftment was observed prior to mobilization (before day 6), during a period of active hematopoietic regeneration following the administration of cyclophosphamide. Hematopoietic regeneration was evidenced by a reduced but rapidly increasing marrow cellularity and an increased proportion of hematopoietic progenitors in S-phase. In contrast, long-term syngeneic donor engraftment was not increased over controls during the period of maximum progenitor and stem cell mobilization (after day 5). At this time there were minimal numbers of progenitor and stem cells in the marrow. These data suggest that in the absence of host stem cell ablation, maximal engraftment does not occur during marrow progenitor or stem cell depletion, suggesting that the presence of "open" marrow sites is not a prerequisite for engraftment. The mechanisms for increased engraftment during progenitor cell regeneration following cyclophosphamide need further investigation. Understanding the mechanisms for engraftment without host stem cell ablation may allow strategies for improved long-term engraftment of syngeneic or autologous stem cells with reduced post-transplant toxicity.     

Successful allogeneic bone marrow transplantation (BMT) by injection of bone marrow cells via portal vein: stromal cells as BMT-facilitating cells

We examined the importance of the coadministration of bone marrow (BM) stromal cells with BM cells via the portal vein. A significant increase in the number of day-14 colony-forming unit-spleen (CFU-S) was observed in the recipient mice injected with hemopoietic stem cells (HSCs) along with donor BM stromal cells obtained after three to four weeks of culture. Histological examination revealed that hematopoietic colonies composed of both donor hemopoietic cells and stromal cells coexist in the liver of these mice. However, when donor HSCs plus BM stromal cells were administered i.v., neither the stimulatory effects on CFU-S formation nor the hemopoietic colonies in the recipient liver were observed. These findings suggest that the interaction of HSCs with stromal cells in the liver is the first crucial step for successful engraftment of allogeneic HSCs. It is likely that donor stromal cells and HSCs trapped in the liver migrate into the recipient BM and spleen, where they form CFU-BM and CFU-S, respectively.     

Optical imaging of PKH-labeled hematopoietic cells in recipient bone marrow in vivo

This work describes an optical technique for characterization of the early stages of hematopoietic stem cell (HSC) engraftment under physiological conditions and in real time. Bone marrow cells (BMCs) labeled with PKH membrane linkers were injected into conditioned recipients (B10-->B10.BR mice) preoperated for placement of optical windows over femoral epiphyses. Labeled cells were tracked in vivo by fluorescence microscopy. Cellular adhesion to the BM stroma was tested with laser tweezers, and viability was assayed by the propidium iodide (PI) exclusion test, as determined from energy-transfer measurements of the pair PKH67-PI in freshly excised femurs in situ. At optimal concentrations for in vivo tracking, 1-4 micro M PKH dyes neither impaired the viability of BMCs nor the capacity of allogeneic HSCs to reconstitute hematopoiesis in myeloablated recipients. The optical window allowed in vivo visualization of 23%-26% of the PKH-labeled BMCs in the femur. The homing efficiencies at 16 hours posttransplantation were quantified as 1.77% +/- 0.15% and 0.21% +/- 0.02% for syngeneic and allogeneic BMCs, respectively. In femurs excised 16 hours after transplantation, 70% +/- 9% of the cells were adherent to the BM stroma, and two-thirds of the cells were PI negative (viable). In vivo tracking and in situ assessment of labeled HSCs in recipient BM provide important quantitative and qualitative insights into the early stages of engraftment. Correlation of early events and the efficiency of durable engraftment serve as the basis for a systematic approach toward optimization of the conditions for transplantation.     

Attenuation of murine lysosomal storage disease by allogeneic neonatal bone marrow transplantation using costimulatory blockade and donor lymphocyte infusion without myeloablation

Treatment of nonmalignant childhood disorders by bone marrow transplantation (BMT) is limited by toxicity from preparatory regimens and immune consequences associated with engraftment of allogeneic donor cells. Using costimulatory blockade (anti-CD40L mAb and CTLA-4Ig) combined with high-dose BMT in nonablated neonates, we obtained engraftment and established tolerance using both partially MHC mismatched (H2g7 into H2b) and fully mismatched BM (H2s into H2b). Recipients were mucopolysaccharidosis type VII (MPS VII) mice with lysosomal storage disease in order to assess therapeutic outcome. Recipients treated with donor lymphocyte infusion (DLI) amplified microchimerism to full donor. Recipients without DLI maintained long-term engraftment, tolerance, and had extended life spans. DLI increased donor cell mediated replacement of beta-glucuronidase (GUSB) activity in all tissues and maintained clearance of lysosomes better than in non-DLI-treated mice. DLI amplification of partially mismatched BM and fully mismatched BM caused late onset chronic GvHD in 56% and 100% of recipients, respectively.