Publications (9)24.95 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: A marker for hematopoietic stem cells (HSCs) of pigs, which are considered to be the most suitable donors for clinical xenotransplantation, has not yet been identified. In this study, we examined the HSC activity of porcine c-kit+ bone marrow cells (BMCs). The HSC activity of porcine c-kit+ BMCs was evaluated both in vitro using colony-forming unit (CFU) and cobblestone area-forming cell (CAFC) assays and in vivo in nonobese diabetic/severe combined immunodeficiency transgenic (NOD/SCID-Tg) mice carrying porcine cytokine transgenes. Purified c-kit+ BMCs were substantially enriched for both CFUs and CAFCs in vitro and their transplantation led to long-term porcine hematopoiesis in vivo in mice. Although porcine chimerism was detectable in the peripheral blood of NOD/SCID-Tg mice receiving porcine c-kit- BMCs at early time points after transplantation, the levels were markedly lower than those in mice receiving purified c-kit+ BMCs (0.2%+/-0.14% vs 7.7%+/-1.6% and 0.17%+/-0.17% vs 5.6%+/-2.1% at weeks 3 and 6, respectively). Importantly, all mouse recipients of porcine c-kit+ BMCs showed durable multilineage chimerism (>19 weeks), whereas no recipients of porcine c-kit- BMCs sustained long-term engraftment. Moreover, porcine HSCs that had engrafted for 19 weeks in the recipients of porcine c-kit+ BMCs gave rise to clonogenic progenitors in vitro and reconstituted porcine hematopoiesis in secondary recipients. The present study demonstrates that c-kit is an essential marker of both long-term-repopulating HSCs and progenitor cells with early engraftment capacity.
- [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to develop novel markers for enrichment of hematopoietic progenitors from bone marrow of swine. We previously showed that pig bone marrow contains a "side population" (SP) of Hoechst dye-effluxing cells that resembles the hematopoietic stem cell (HSC)-containing murine SP and therefore represents a putative pig stem cell population. We screened a panel of monoclonal antibodies for those that allowed positive or negative enrichment of porcine SP cells and tested one of these for enrichment of hematopoietic progenitors in short-term and long-term in vitro assays. We then screened an expression library to clone the gene whose product is recognized by this antibody. Among a panel of 35 monoclonal lines screened, we found three that were useful for positive enrichment of SP cells and seven for negative enrichment. The 4-6 monoclonal line, allowing around 10-fold negative enrichment of SP cells, recognized the product of the porcine CD9 gene. Hematopoietic progenitors measured by short-term colony-forming unit and long-term cobblestone area-forming cell assays were around 10-fold enriched in the CD9(negative/low) fraction and were significantly depleted in the CD9(high) fraction. The antibody against the porcine CD9 gene product may be of use for enrichment of porcine hematopoietic stem cells. This approach to identify novel markers for enrichment of hematopoietic progenitors may be applicable to other mammalian species.
- [Show abstract] [Hide abstract] ABSTRACT: To combat the shortage of donor organs, transplantation across species barriers has been proposed. Induction of tolerance would overcome the substantial immunologic barriers to xenotransplantation and would avoid the chronic use of immunosuppressive agents. Successful transplantation of hematopoietic cells induces robust specific tolerance to donor antigens in allogeneic and xenogeneic models. The beta1 integrin class of adhesion molecules and their interactions with extracellular matrix components are thought to be integral to the engraftment and maturation of hematopoietic stem cells. We therefore examined the efficacy of porcine very late antigen-5 (VLA-5) and VLA-4 interactions with the human extracellular matrix (ECM) protein, fibronectin. Peripheral blood mononuclear cells (PBMCs) from humans and miniature swine were flourochrome labeled and adhesion to plates coated with whole human fibronectin (whFN) or its 120 KDa fragment containing the VLA-5 binding region was determined. Flow cytometry and immuno- precipitation were used to identify a monoclonal antibody that cross-reacted on porcine VLA-5. Human and pig PBMC adhesion to human fibronectin (hFN) or 120 kDa fragment-coated plates was assessed following incubation with control ab, anti-VLA-4, anti-VLA-5, or soluble fibronectin. Using rabbit complement, cells expressing VLA-5 were purged from PBMC preparations before performing the adhesion assay. Porcine and human PBMC both adhered to hFN in a divalent cation-dependent and activation-dependent manner. Adhesion to hFN of human but not pig PBMC was blocked by anti-VLA-5 monoclonal antibody SAM-1, although this mAb immunoprecipitated a heterodimeric cell surface molecule (155/135 kDa) resembling VLA-5 from pig PBMC. Complement-mediated depletion of VLA-5-expressing cells ablated specific binding of human but not porcine cells to hFN and its 120 kDa fragment. Addition of soluble fibronectin was capable of blocking adhesion of PBMC of both species to hFN. Anti-VLA-4 reduced the binding of PBMC from both species to hFN to a similar extent. Human and pig cells can specifically adhere to hFN and its 120 kDa fragment, suggesting that this critical cell-ECM interaction is preserved across species. While human cells exclusively use VLA-5 for binding to the 120 kDa fragment, porcine cells could not be shown to adhere to whFN or its 120 kDa fragment via VLA-5. However, porcine VLA-4 is capable of mediating adhesion to human FN. We conclude that disparities in the adhesive interactions of beta1 integrins may be a barrier to the use of porcine hematopoietic stem cell transplantation as a means of inducing donor-specific tolerance in the pig to human species combination.
- [Show abstract] [Hide abstract] ABSTRACT: The molecular interactions of intercellular adhesion molecule-1 (ICAM-1; CD54) are potentially important in several situations in the context of pig-to-human xenotransplantation. If porcine bone marrow is to be used for the induction of xenograft tolerance in humans, the role that has been suggested for ICAM-1 in the interactions of haematopoietic stem cells makes its cross-species compatibility important. Similarly, the potential role of ICAM-1 interactions in graft rejection makes it an important molecule to study. An in vitro static cell-to-cell adhesion study was used to look at the successful interaction of ICAM-1 with its ligands across the pig-human species barrier in both directions. A second in vitro system, the standard long-term bone marrow culture (LT-BMC), was used to study the functional role of ICAM-1 in haematopoiesis. Human ICAM-1 was able to adhere to ligands on porcine cells, including one or more ligand that contains CD18. Conversely, human CD18-containing ligands mediated adherence to porcine cells. Using the long-term bone marrow culture system, there was no evidence that blocking the interactions of ICAM-1 inhibited hematopoiesis, either in the human-human or pig-human combinations of precursor cells and marrow stroma. ICAM-1 is able to interact with at least some of its ligands across the species barrier, in both pig-human and human-pig combinations. However, the interactions of ICAM-1 do not appear to be central to hematopoiesis, at least in the model system used.
- [Show abstract] [Hide abstract] ABSTRACT: The capacity of mixed hematopoietic chimerism to induce tolerance has not been demonstrated in discordant xenogeneic species combinations because of the difficulty in achieving lasting hematopoietic engraftment. In an effort to create a model of long-lasting disparate xenogeneic hematopoietic chimerism, we have developed transgenic (Tg) mice carrying porcine cytokines. Three lines of Tg mice were generated: one carrying porcine IL-3 and GM-CSF genes only (termed IL/GM) and the remaining two lines carrying in addition, the soluble SCF gene (termed IL/GM/sS) or membrane-bound SCF gene (termed IL/GM/mS). Sera from mice with IL/GM and IL/GM/sS transgenes markedly stimulated the proliferation of swine marrow cells in vitro. However, proliferation of swine marrow cells was not induced in cultures containing IL/GM/mS sera. Consistent with these observations, ELISA assays revealed detectable levels of porcine cytokines in the sera of IL/ GM and IL/GM/sS, but not in sera of IL/GM/mS Tg mice. Marrow stromal cells prepared from all three kinds of Tg mice, but not those from non-Tg littermates, were capable of supporting the growth of porcine hematopoietic cells in vitro. Immunodeficient Tg mice were generated by crossing Tg founders with C.B-17 SCID mice for five generations. All Tg immunodeficient mice showed improved porcine hematopoietic engraftment compared with non-Tg controls. These Tg mice provide a useful model system for studying porcine hematopoietic stem cells, and for evaluating the feasibility of donor-specific tolerance induction by mixed chimerism across highly disparate xenogeneic barriers.
- [Show abstract] [Hide abstract] ABSTRACT: Background. The molecular interactions of intercellular adhesion molecule-1 (ICAM-1; CD54) are potentially important in several situations in the context of pig-to-human xenotransplantation. If porcine bone marrow is to be used for the induction of xenograft tolerance in humans, the role that has been suggested for ICAM-1 in the interactions of haematopoietic stem cells makes its cross-species compatibility important. Similarly, the potential role of ICAM-1 interactions in graft rejection makes it an important molecule to study. Methods. An in vitro static cell-to-cell adhesion study was used to look at the successful interaction of ICAM-1 with its ligands across the pig-human species barrier in both directions. A second in vitro system, the standard long-term bone marrow culture (LTBMC), was used to study the functional role of ICAM-1 in haematopoiesis. Results. Human ICAM-1 was able to adhere to ligands on porcine cells, including one or more ligand that contains CD18. Conversely, human CD18-containing ligands mediated adherence to porcine cells. Using the long-term bone marrow culture system, there was no evidence that blocking the interactions of ICAM-1 inhibited hematopoiesis, either in the human-human or pig-human combinations of precursor cells and marrow stroma. Conclusions. ICAM-1 is able to interact with at least some of its ligands across the species barrier, in both pig-human and human-pig combinations. However, the interactions of ICAM-1 do not appear to be central to hematopoiesis, at least in the model system used.
- [Show abstract] [Hide abstract] ABSTRACT: Immune responses to xenografts are likely to be highly dependent on the efficiency of molecular interactions between the donor and the recipient species. This brief review summarizes what is currently known about the compatibilities across the human-porcine species barrier of the molecular interactions that are important in the immune response.
- [Show abstract] [Hide abstract] ABSTRACT: Background. One way to circumvent the need for chronic immunosuppression in solid organ xenografting may be to induce donor-specific tolerance using bone marrow transplantation. If this approach is to succeed in the pig-to-human species combination, pig marrow must be capable of maturing into relevant tolerance-inducing cells and replenishing itself in host human marrow. One possible barrier is adhesion molecule incompatibility. We have studied the compatibility across the pig-human species barrier of two well-characterized ligands known to be important in hematopoiesis, CD44 and very late antigen(VLA)-4. Methods. In vitro long-term bone marrow cultures were studied in which the effects of blocking antibodies were assessed by measuring cell numbers and colony-forming units. Results. The blocking of CD44 had a comparable inhibitory effect on the hematopoiesis of human and pig marrow, even if the latter was maintained on a human stromal layer. Both cellular proliferation and colony-forming activity were inhibited by anti-CD44 monoclonal antibody. By contrast, a significant difference was observed in VLA-4 usage by hematopoietic cells of the two species. Blocking VLA-4 markedly inhibited human hematopoietic cellular proliferation but had no effect on pig hematopoiesis, on either porcine or human stroma. Conclusion. The data suggest that the incompatibility of either CD44 or VLA-4 is unlikely to limit the efficiency of porcine hematopoiesis in a human marrow environment. However, the difference in VLA-4 utilization between these species raises the possibility that other interactions may be important for effective porcine hematopoiesis and that their failure to function between species may contribute to the poor function of porcine hematopoietic cells in primate marrow microenvironments.
- [Show abstract] [Hide abstract] ABSTRACT: Induction of donor-specific tolerance via bone marrow transplantation has been proposed as a means of overcoming immunologie barriers to xenotransplaniaiion Bone marrow transplantation is dependent on a complex set of adhesive interactions permitting engraftmem, maturation and migration of donor hemalopoielic cells. Thtintegrtn class of adhesion molecules has been demonstrated to be of significance in human and mouse bone marrow transplantation. The purpose of this study was to asses the function of the : integrin VLA-5 via long term bone marrow culture (LTBMC) and static adhesion studies in the pig to human xenogenic combination Human and pig bone marrow stromal layers were cultured and seeded with fresh hematopoietic precursor cells from pig or human bone marrow specimens. Culture cell counts and colony forming assays (CPU) were performed on a weekly basis. A mouse anti-human VLA-5 monoclonal antibody SAM-1, demonstrated by imrnunoprecipitation to be cross-reactive on the porcine molecule, was added to cultures and compared to a control antibody for effects on cellular proliferation and CPU potential. Similar proportions of porcine and human bone marrow cells showed expression of VLA-5 and the binding titer was similar in both species. Hematopoietic proliferation of human cells and CPU development were significantly impaired by the addition of anti-VLA-5 mAb to LTBMC. In contrast, neither hematopoietic cell proliferation nor CPU development of porcine LTBMC were impaired by anti VLA5 mAb. In static adhesion assays, human and porcine leukocytes labeled with calcein were assessed for adhesion to human fibronectin (a principal ligand for VLA-5 ). Monoclonal antibodies directed against VLA-5 were added to assays and compared to control antibody for the ability to block VLA-5- mediated adhesion to fibronectin. Although both porcine and human cells adhered to human fibronectin, the adhesion of human cells to fibronectin-coated plates was impaired by anri-VLA-5 mAB, but that of pig cells was not. The differential blocking effects of anti-VLA-5 mAb on hematopoiesis and adhesion to human fibronectin of pig versus human cells suggests that VLA-5 function differs in a species-specific manner, or that the SAM-1 mAb recognizes a non- functional porcine VLA-5 epitope. Studies are in progress to investigate these possibilities.
Mass General HospitalBoston, Massachusetts, United States
Massachusetts General Hospital
Boston, Massachusetts, United States
- Transplantation Biology Research Center