Generation of vascular chimerism within donor organs.

Whole organ perfusion decellularization has been proposed as a promising method to generate non-immunogenic organs from allogeneic and xenogeneic donors. However, the ability to recellularize organ scaffolds with multiple patient-specific cells in a spatially controlled manner remains challenging. Here, we propose that replacing donor endothelial cells alone, while keeping the rest of the organ viable and functional, is more technically feasible, and may offer a significant shortcut in the efforts to engineer transplantable organs. Vascular decellularization was achieved ex vivo, under controlled machine perfusion conditions, in various rat and porcine organs, including the kidneys, liver, lungs, heart, aorta, hind limbs, and pancreas. In addition, vascular decellularization of selected organs was performed in situ, within the donor body, achieving better control over the perfusion process. Human placenta-derived endothelial progenitor cells (EPCs) were used as immunologically-acceptable human cells to repopulate the luminal surface of de-endothelialized aorta (in vitro), kidneys, lungs and hind limbs (ex vivo). This study provides evidence that artificially generating vascular chimerism is feasible and could potentially pave the way for crossing the immunological barrier to xenotransplantation, as well as reducing the immunological burden of allogeneic grafts.

Stable long-term mixed chimerism achieved in a canine model of allogeneic in utero hematopoietic cell transplantation.

Evidence supporting the efficacy of in utero hematopoietic cell transplantation (IUHCT) in a valid large animal model is needed prior to clinical application. The objective of this study was to establish clinically relevant levels of hematopoietic chimerism in a canine model of maternal-to-fetal IUHCT. We first assessed immune and hematopoietic ontogeny relevant to IUHCT in the canine model and identified 40 days' gestation (term 63 days) as a time point at the initiation of thymic selection, and prior to bone marrow hematopoiesis, that might be optimal for IUHCT. We next determined that intravascular administration of donor cells via intracardiac injection was far more efficient and resulted in much higher levels of donor cell engraftment than intraperitoneal injection. By applying these findings, we achieved stable long-term multilineage engraftment in 21 of 24 surviving recipients with an average level of initial chimerism of 11.7% (range 3% to 39%) without conditioning or evidence of graft-versus-host disease. Donor cell chimerism remained stable for up to 2 years and was associated with donor-specific tolerance for renal transplantation. The levels of donor cell chimerism achieved in this study would be therapeutic for many hematopoietic disorders and are supportive of a clinical trial of IUHCT.

Post transplant persistence of host cells augments the intensity of acute graft-versus-host disease and level of donor chimerism, an explanation for graft-versus-host disease and rapid displacement of host cells seen following non-myeloablative stem cell transplantation?

Although the use of non-myeloablative stem cell transplantation (NST) reduces the severity of graft-versus-host disease (GVHD), GVHD remains a major complication following allogeneic transplantation. Since following NST in comparison with myeloablative conditioning, higher proportions of host immunohematopoietic cells may persist while donor-derived alloreactive lymphocytes are being infused, thus possibly serving as host antigen presentation for continuous stimulation of donor T cells, we speculated that GVHD may be similarly amplified by conditioning followed by intentional administration of host cells. This hypothesis was tested in a preclinical animal model. Increased incidence of GVHD, higher mortality and increased levels of chimerism were observed in recipients reconstituted with host cells, particularly with non-irradiated spleen cells. Graft-versus-leukemia effect was not impaired by post transplant cell administration. These results suggest that GVHD may be amplified by recipient cell infusion using either irradiated or viable stimulatory host cells, thus possibly explaining in part higher than anticipated incidence of GVHD and rapid displacement of host cells and conversion to 100% donor type cells following NST. Administration of irradiated host antigen-presenting cells post transplantation may thus represent a potential approach for amplification of the alloreactive capacity of donor lymphocytes following stem cell transplantation.

Pluripotency of cryopreserved blastomeres of the goldfish.

To examine the pluripotency of cryopreserved blastomeres, we transplanted them into blastula. Donor blastomeres were prepared from blastula of goldfish (Carassius auratus) and cryopreserved in liquid nitrogen for two months. Fifty-five percent and 44% of blastomeres survived after thawing. Cryopreserved blastomeres were transplanted to the blastula of triploid crucian carp (C. a. longsdorfii), which reproduces gynogenetically in nature. At four days after the operation, resultant chimeric embryos transplanted with cryopreserved blastomeres showed a survival rate (41.6%) lower than that of embryos transplanted with unfrozen blastomeres (57.1%). Transplanted blastomeres were histologically identified in various organs derived from all three germ layers. A primordial germ cell differentiated from a cryopreserved blastomere was detected in one of the 32 chimeric fish examined. These results suggest blastomeres that survive after cryopreservation retain their pluripotency and are able to differentiate into both somatic and germ cell lines.

Host cell-derived cardiomyocytes in sex-mismatch cardiac allografts.

BACKGROUND: Mesenchymal precursor cells are able to respond to tissue signals and differentiate into a phenotype characteristic of mature cells of that tissue. We sought to investigate whether adult human cardiomyocytes can be derived from recipient precursor cells in sex-mismatched cardiac allografts. METHODS: We studied four male patients who received hearts from female donors, and four female patients who received an allograft from a male donor. Four sex-matched transplant patients, two of each sex served as controls. Combined fluorescence in situ hybridization with probes specific for X- and Y-chromosomes and immunohistochemistry with alpha-actin was used to identify cardiac muscle cells 4 and 12 months after transplantation. Slides were examined with a fluorescence microscope to detect the presence of male cells with one X and one Y signal in the nucleus, and female cells containing two X signals. RESULTS: Mature cardiomyocytes from the host (1-2%) were found in five endomyocardial biopsy specimens at 4 months, and in three specimens at 12 months. In addition, recipient cells negative for cytoplasmic alpha-actin were also identified (1-21% per slide). The number of infiltrating recipient cells was not associated with the degree of rejection of the sample or with the number of prior rejection episodes. Echocardiographic evaluation showed no improvement in cardiac performance in hearts from patients with more than 10% chimeric recipient cells. CONCLUSIONS: Our data confirm the existence of mature cardiomyocytes derived from host cells, likely mesenchymal precursors, in the adult cardiac allograft in vivo.

Facial visceral motor neurons display specific rhombomere origin and axon pathfinding behavior in the chick.

In the chick embryo, facial motor neurons comprise branchiomotor and visceral motor subpopulations, which innervate branchial muscles and parasympathetic ganglia, respectively. Although facial motor neurons are known to develop within hindbrain rhombomere 4 (r4) and r5, the precise origins of branchiomotor and visceral motor neuron subpopulations are unclear. We investigated the organization and axon pathfinding of these motor neurons using axonal tracing and rhombomere transplantation in quail-chick chimeras. Our results show that a large majority of branchiomotor neurons originate in r4 but that a cohort of these neurons undergoes a caudal migration from r4 into r5. By contrast, visceral motor neurons develop exclusively in r5. We found that a striking property of facial visceral motor neurons is the ability of their axons to navigate back to appropriate ganglionic targets in the periphery after heterotopic transplantation. These results complement previous studies in which heterotopic facial branchiomotor neurons sent axons to their correct, branchial arch, target. By contrast, when trigeminal branchiomotor neurons were transplanted heterotopically, we found that they were unable to pathfind correctly, and instead projected to an inappropriate target region. Thus, facial and trigeminal motor neuron populations have different axon pathfinding characteristics.

Production of chicken chimeras by fusing blastodermal cells with electroporation.

AIM: To establish techniques for producing somatic and germline chimeric chicken by transferring blastodermal cells fused with electroporation. METHODS: Stage-X blastodermal cells isolated from freshly laid fertile unincubated white Leghorn and Rhode Island red chicken eggs were fused with electroporation. The treated cell suspension was transferred to the recovery medium (DMEM containing 10% FBS) and was injected into the subgerminal cavity of recipient unincubated embryos (stage X). RESULTS: Of 177 recipient embryos injected with the fusing blastodermal cells, 6 (3.4%) survived to hatching. Somatic chimerism was examined in the melanocyte of the feather. The presence of feathers originating from the donor cell was observed in 1 bird (16.7%) out of the 6 hatched birds. After 21 days of incubation two birds out of five embryos were subjected to polymerase chain reaction (PCR) analysis for W-chromosome-specific DNA for each tissue. One bird possessed W-chromosome-specific DNA in the stomach, and the other exhibited the same DNA in the left and right gonads and other tissues, but not the stomach. CONCLUSION: Recipient embryo having electrofused blastodermal cells yields somatic and germline chimeric chickens more successfully.