Cells Of Host Origin Research Articles

The regenerative treatment of costal osteochondral graft implantation for partial growth arrest using a rabbit model

PurposeTo investigate the feasibility of cylindrical costal osteochondral graft transplantation as a novel regenerative treatment in growth arrest. MethodsThe medial portion of the proximal tibial growth plate of 6-week-old male New Zealand White rabbits was resected to establish an experimental model of partial growth plate injury. The rabbits were divided into four groups: no-treatment, bone wax transplantation, costal chondral graft, and costal osteochondral graft groups. Radiographic and micro-computed tomography scan results were analyzed to evaluate angular deformity of the tibia and bony bridge formation at the injury site. In addition, repair of the injured growth plate cartilage was assessed histologically at 4, 8, and 12 weeks postoperatively. ResultsRadiographic examination revealed that bone wax transplantation continuously decreased the medial proximal tibial angle (MPTA) while the costal chondral graft implantation reduced the decrease of MPTA at 12 weeks postoperatively. The costal osteochondral graft implantation recovered the MPTA, close to the normal. Histologically, the costal osteochondral grafts retained the MPTA in the injured site compared to costal chondral grafts. Additionally, hypertrophic chondrocytes were observed at the graft site in the costal osteochondral graft group at 12 weeks, suggesting that endochondral ossification may occur at the graft site similar to normal ossification. The fluorescence in situ hybridization analysis of osteochondral grafts transplanted from male to female rabbits indicated that they were replaced by cells of host origin. ConclusionThe costal osteochondral graft can achieve regeneration without bony bridge formation in partial growth plate injury.

Artificial collagen-filament scaffold promotes axon regeneration and long tract reconstruction in a rat model of spinal cord transection.

Traumatically injured spinal cord (SC) displays structural damage that includes discontinuity of long tracts and cavitations. Axonal regrowth beyond the lesion is necessary to achieve functional recovery following SC injury. We report here the development of an artificial collagen-filament (CF) scaffold to replace the SC in 8-week-old female Fisher rats. Axonal sprouting and regrowth was very rapid following grafting of the CF. One week after implantation, the scaffold was filled with cells of host origin and with regenerated axons. Histological examination of SC adjacent to the scaffold showed little cavity formation or fibrous scarring. Eight weeks after implantation, myelinated nerve fibers were found in the scaffold and 10-25% of rubrospinal tracts were repaired. Four to six weeks after transplantation, motor evoked potentials were recorded in CF-grafted rats but were not detectable in non-grafted rats. Electrophysiological and histological examinations revealed the grafted CF was likely to function as a nerve tract. In addition, these results suggest that collagen fibers may provide a permissive microenvironment for the elongation of SC axons and to support the process of spinal cord regeneration.

Integrating repopulation and regeneration of the auxiliarily transplanted small liver graft: The solution for organ shortage and immunosuppression

Cirrhosis is a common yet generally irreversible medical condition which would cause damaged liver function and regeneration after resection. When the disease progressed to end stage, liver transplantation, cadaveric or live donor, would be the only way to eliminate the disease. But both have their confinements, such as organ shortage, detriments to the donors, and immunosuppression.Now that the techniques of auxiliary transplantation are in practice, and the phenomenon of graft repopulated by cells of host origin has been observed, and that elevated blood inflow would stimulate the regenerative response, the combination of the three might give rise to a non-immunogeneic customized liver. We hypotheses that it can be achieved through a auxiliary transplantation of a extremely small but normal graft combined with progressive portal control on the portal inflow of the native liver to induce atrophy of the cirrhotic liver and the regeneration of the graft, as well as postoperative administration of bone marrow mobilizing agents and reduced administration of immunosuppressants to initiate repopulation.This will not only solve the issue of organ shortage as one organ can be shared by more, and in case of live donor, less detriments would occur due to reduced size needed; but diminish, even eliminate the adverse effect caused by immunosuppression as well.

Neural precursor cells differentiated from mouse embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson’s disease

Pluripotent embryonic stem (ES) cells are the most versatile cells, with the potential to differentiate into all types of cell lineages including neural precursor cells (NPCs), which can be expanded in large numbers for significant periods of time to provide a reliable cell source for transplantation in neurodegenerative disorders such as Parkinson’s disease (PD). In the present study, we used the MESPU35 mouse ES cell line, which expresses enhanced green fluorescent protein that enables one to distinguish between transplanted cells and cells of host origin. Embryoid bodies (EBs) were formed and were induced to NPCs in N2 selection medium plus fibronectin. Praxiology and immunohistochemistry methods were used to observe the survival, differentiation, and therapeutic effect of NPCs after grafted into the striatum of PD rats. We found that mouse ESc were differentiated into nestin-positive NPCs 6 days after the EBs formed and cultured in the N2 selection medium. The number of survival NPCs was increased significantly by fibronectin. About 23.76 ± 2.29% of remaining cells were tyrosine hydroxylase (TH)-positive 12 days after NPCs were cultured in N2 selective medium. The survival rates of NPCs were 2.10 ± 0.41% and about 90.90 ± 3.00% of the engrafted NPCs were TH-positive 6 weeks after transplantation into the striatum of PD rats. The rotation of PD rats was relieved 3 weeks after the NPCs transplantation and this effect was kept for at least 6 weeks. It suggests that most of the survival NPCs derived from ES cells differentiated into TH-positive neurons after grafted into the striatum of PD rats, which produces therapeutic effect on PD.

Dynamics of medial smooth muscle changes after carotid artery transplantation in transgenic mice expressing green fluorescent protein.

Recent observations have demonstrated the importance of host cells in neointima formation after transplantation. Because little is known regarding the dynamics of host-derived cells in the graft media, we investigated this question in a mouse carotid artery transplantation model. C57BL/6 carotid arteries were orthotopically transplanted into BALB/c mice ubiquitously expressing enhanced green fluorescent protein. Grafts were harvested at 1, 2, 4, and 8 weeks after transplantation for histologic examination. No immunosuppression was used. Immunostaining and semiquantitative analysis of cross sections showed that donor medial smooth muscle cells decreased over time in the graft media, whereas green fluorescent protein-positive/smooth muscle alpha-actin-positive cells (i.e., cells of host origin) increased over time. Interestingly, host cells were located only in the inner media and the neointima at 2 weeks and thereafter also in the outer media, indicating that the host-derived cells entered the media from the luminal side rather than from the adventitia. In longitudinal sections, there were no differences in the accumulation of donor- and host-derived cells between the end and middle regions of the graft media at 8 weeks. After transplantation, medial cells were replaced by alpha-actin-expressing host cells that were probably derived from circulating precursor cells. Our observations differ from the traditional view of a major contribution of donor medial smooth muscle cells to the neointima formation. Thus, circulating progenitor cells may be important for graft vessel disease.

Marrow stroma in MDS: culprit or bystander?

The marrow microenvironment consists of numerous components, including fibroblast-like cells, adipocytes, endothelial cells, smooth muscle cells, macrophages and others [1,2]. At times the term stroma is used interchangeably with microenvironment, although others, including myself, prefer to use that term only when talking about adherently growing fibroblast-like cells as recognized, e.g. by the monoclonal antibody STRO-1 [3]. Results of marrow and peripheral blood stem cell transplants in animal models and humans, have shown that following successful transplantation, macrophages are replaced by cells of donor origin, whereas fibroblast-like (stroma) cells remain of host origin [4,5]. The position of endothelial cells is somewhat controversial, as it was shown that vascular prostheses became lined by endothelial cells of donor origin in recipients given an allogeneic haemopoietic stem cell transplant [6]. Patients with a myelodysplastic syndrome (MDS) can be cured by allogeneic (and in some instances possibly even by autologous) haemopoietic stem cell transplantation. This suggests that the microenvironment and in particular the stroma of these patients either is intact or if alterations are present, they are likely to be reversible. Alternatively, a (small) subpopulation of stroma cells may indeed be part of MDS but is eliminated by successful haemopoietic stem cell transplantation [7]. Residual normal stroma (or mesenchymal) stem cells of host origin would then replenish the (host) stromal components following transplantation [8]. In this issue of Leukemia Research, Flores-Figueroa et al. report that fibroblasts derived from MDS marrow, produce significantly higher levels of interleukin-6 (IL-6) and tumor necrosis factor(TNF), and MDS macrophages produce significantly higher levels of TNFthan do their normal counterparts [9]. The macrophage data are not surprising. They confirm earlier reports [2,10] and are consistent with the fact that these cells are derived from haemopoietic stem cells [11,12]. More intriguing, albeit not without

Maternal-fetal relationship, natural chimerism and bilateral transplantation tolerance as the basis for non-myeloablative stem cell transplantation.

Bone marrow transplantation (BMT) which represents an important clinical tool for treatment of patients with a wide variety of malignant and non-malignant diseases, however, the procedure is associated with procedure-related toxicity and mortality as well as unavoidable late complications. Many of the undesirable consequences of BMT are caused directly or indirectly by the intensive conditioning administered during the pre-transplant period. However, if the main goal of the BMT procedure is to enable immunotherapy by alloreactive donor lymphocytes, the conditioning prior to BMT needs to be reconsidered, because transplantation tolerance across major histocompatibility complex (MHC) occurs spontaneously in nature, as evidenced by the fact that pregnant females do not reject their conceptus. In fact, as shown by Owens in the 1940s, placental parabiosis in utero leads to permanent mixed chimerism and bilateral transplantation tolerance. These observations followed by experiments carried out in the 1950s by Billingham et al. suggested that infusion of parental stem cells into neonates with no exogenous immunosuppressive treatment resulted in mixed chimerism and permanent transplantation tolerance to donor alloantigens. Thus, a window of opportunity provided shortly after delivery, was sufficient for induction of tolerance without the need for heavy conditioning. Tolerant recipients were shown to be chimeras with only a small proportion of donor cells. However, without corroborating evidence that transplantation tolerance could be intentionally induced, the approach could not be applied in clinical practice for immunocompetent recipients. Starting in 70s, we documented the feasibility of establishing bilateral transplantation tolerance by mixed chimerism following non-myeloablative conditioning in immu. nologically mature recipients across MHC in mice, rats and dogs. Several studies have shown that reduced intensity conditioning can be very useful for immunoregulation whereas more intensive the pre-grafting immunosuppression resulted in more aggressive the GVHD. These and other findings suggested that lower intensity conditioning may be sufficient for engraftment of donor stem cells, thus suggesting that immunosuppression without myeloablation may be sufficient for prevention of allograft rejection. Following engraftment of donor stem cells, donor lymphocytes infused with bone marrow or mobilized blood stem cells can eradicate residual hematopoietic cells of host origin, occasionally non-hematopoietic tumor cells of host origin as well. Whenever indicated, donor lymphocytes infusion (DLI) can be used at a later stage post BMT to eradicate residual malignant cells of host origin or for the treatment of residual or recurrent disease. Taken together, ongoing clinical studies suggest that high-dose, myeloablative chemoradiotherapy, could be safely replaced with non-myeloablative conditioning (NST).

Treatment of leukemia by alloreactive lymphocytes and nonmyeloablative stem cell transplantation.

Allogeneic bone marrow or blood stem cell transplantation (BMT) represents an important therapeutic tool for treatment of otherwise incurable malignant and nonmalignant diseases, especially acute and chronic leukemias. Until recently, myeloablative regimens were considered mandatory for effective eradication of all malignant cells of host origin. Our preclinical and ongoing clinical studies indicated that eradication of host immunohematopoietic cells, including chemoradiotherapy-resistant leukemia, could be achieved by adoptive allogeneic cell therapy with donor lymphocyte infusion following induction of host-versus-graft transplantation tolerance mediated by engraftment of donor stem cells in the course of BMT. Thus, eradication of blood cancer cells, especially in patients with chronic myeloid leukemia and less frequently in patients with other hematologic malignancies, could be frequently accomplished despite complete resistance of such tumor cells to maximally tolerated doses of chemoradiotherapy. Our cumulative experience suggested that graft-versus-leukemia (GVL) effects might be a useful tool for both treatment and prevention of relapse. Based on the aforementioned rationale, we speculated that the therapeutic benefit of BMT may be improved by using a safer conditioning as part of the transplant procedure, with the goal in mind to induce host-versus-graft tolerance to enable subsequent induction of GVL effects rather than attempt to eliminate host cells with hazardous myeloablative chemoradiotherapy. The latter hypothesis suggested that effective BMT procedure may be accomplished without lethal conditioning of the host, using a new well-tolerated nonmyeloablative regimen, thus possibly minimizing immediate and late side effects related to myeloablative procedures considered until recently mandatory for conditioning of BMT recipients. Recent clinical observations suggest that effective treatment of leukemia may be accomplished with a well-tolerated nonmyeloablative stem cell transplantation (NST) regimen, while avoiding immediate and late toxicity and minimizing procedure-related mortality. Taken together, our cumulative data suggest that high-dose chemotherapy and radiation therapy may be successively replaced by a more effective biological tool-alloreactive donor lymphocytes-thus setting the stage for innovative immunotherapeutic procedures for more selective and effective treatment of patients in need of BMT, including those resistant to conventional chemoradiotherapy.

Long-Term Chimerism and B-Cell Function After Bone Marrow Transplantation in Patients With Severe Combined Immunodeficiency With B Cells: A Single-Center Study of 22 Patients

We retrospectively analyzed the B-cell function and leukocyte chimerism of 22 patients with severe combined immunodeficiency with B cells (B(+) SCID) who survived more than 2 years after bone marrow transplantation (BMT) to determine the possible consequences of BMT procedures, leukocyte chimerism, and SCID molecular deficit on B-cell function outcome. Circulating T cells were of donor origin in all patients. In recipients of HLA-identical BMT (n = 5), monocytes were of host origin in 5 and B cells were of host origin in 4 and of mixed origin in 1. In recipients of HLA haploidentical T-cell-depleted BMT (n = 17), B cells and monocytes were of host origin in 14 and of donor origin in 3. Engraftment of B cells was found to be associated with normal B-cell function. In contrast, 10 of 18 patients with host B cells still require Ig substitution. Conditioning regimen (ie, 8 mg/kg busulfan and 200 mg/kg cyclophosphamide) was shown neither to promote B-cell and monocyte engraftment nor to affect B-cell function. Eight patients with B cells of host origin had normal B-cell function. Evidence for functional host B cells was further provided in 3 informative cases by Ig allotype determination and by the detection, in 5 studied cases, of host CD27(+) memory B cells as in age-matched controls. These results strongly suggest that, in some transplanted patients, host B cells can cooperate with donor T cells to fully mature in Ig-producing cells.

Development of mouse embryonic stem (ES) cells: IV. Differentiation to mature T and B lymphocytes after implantation of embryoid bodies into nude mice.

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cels differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies “ES fetuses.” In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combined in vitro and in vivo system are discussed.

Specific destruction of host-reactive mature T cells of donor origin prevents graft-versus-host disease in cyclophosphamide-induced tolerant mice.

In cyclophosphamide (CP)-induced tolerance, a long lasting skin allograft tolerance was established in many H-2-identical strain combinations without graft vs host disease. Destruction of donor-reactive T cells of host origin, followed by intrathymic clonal deletion of these cells, has been revealed to be the chief mechanisms of this system. Here, we studied the fate of host-reactive populations in donor-derived T cells of C3H/He (C3H) (H-2k, Mls-1b, Mls-2a) mice rendered CP-induced tolerant to AKR/J (AKR) (H-2k, Mls-1a, Mls-2b), by assessing AKR-derived Thy-1.1+ T cells bearing TCR V beta 3 that are specifically reactive with Mls-2a-encoded Ag of the recipient C3H mice. In the AKR-derived Thy-1.1+ lymph node cells of the C3H mice that had been treated with AKR spleen cells plus CP, CD4(+)-V beta 3+ T cells were obviously decreased by day 10 after the CP treatment. At this stage, the Thy-1.1+ T cells were not detected in the C3H thymus, suggesting that the obvious decrease of CD4(+)-V beta 3+ T cells of AKR origin was not due to intrathymic clonal deletion in the recipient C3H mice. Therefore, the destruction of the host-reactive mature T cells of donor origin, as well as that of the donor-reactive mature T cells of host origin, occurred by the CP treatment at the induction phase. Furthermore, after the establishment of intrathymic mixed chimerism in the recipient C3H mice, V beta 3+ T cells were not detected among the Thy-1.1+ T cells of AKR origin in the mixed chimeric thymus, suggesting that the host-reactive immature T cells repopulated from the injected donor hematopoietic cells were clonally deleted in the recipient thymus. These two mechanisms appear to prevent graft vs host disease in CP-induced tolerance.

Molecular Analysis of Relapse in Chronic Myeloid Leukemia After Allogeneic Bone Marrow Transplantation

Four patients with Philadelphia (Ph') positive chronic myeloid leukemia (CML) were studied before, after, and on relapse following allogeneic bone marrow transplantation (BMT). Southern analysis of DNA from cells collected before and at relapse after BMT was performed in order to investigate the origin of the leukemia at relapse. Using minisatellite probes we showed that the relapse occurred in cells of host origin in all four patients and this was confirmed with a Y chromosome specific probe in two male patients who had a female donor. Furthermore, using two probes for the breakpoint cluster region (bcr) on chromosome 22, we showed that leukemic cells at relapse bore identical rearrangements to those in the disease at time of presentation of each patient. We conclude that relapse in all four patients is due to re-emergence of the original leukemic clone.

Molecular analysis of relapse in chronic myeloid leukemia after allogeneic bone marrow transplantation

Four patients with Philadelphia (Ph′) positive chronic myeloid leukemia (CML) were studied before, after, and on relapse following allogeneic bone marrow transplantation (BMT). Southern analysis of DNA from cells collected before and at relapse after BMT was performed in order to investigate the origin of the leukemia at relapse. Using minisatellite probes we showed that the relapse occurred in cells of host origin in all four patients and this was confirmed with a Y chromosome specific probe in two male patients who had a female donor. Furthermore, using two probes for the breakpoint cluster region (bcr) on chromosome 22, we showed that leukemic cells at relapse bore identical rearrangements to those in the disease at time of presentation of each patient. We conclude that relapse in all four patients is due to re-emergence of the original leukemic clone.

Clonal Origin of Tumors Induced by Ultraviolet Radiation<xref ref-type="fn" rid="FN2">2</xref>

Skin tumors were induced in (C3H/HeN X C3H/HeN-PGK-1a)F1 female mice, heterozygous at the X-linked phosphoglycerate kinase-1 (PGK-1) locus, by exposure to the carcinogenic influence of ultraviolet radiation (UVR), 3-methylcholanthrene [(MCA) CAS: 56-49-5], or benz[a]pyrene [(BP) CAS: 50-32-8]. An assessment of the clonal origin of these tumors was accomplished through an analysis of the PGK-1 enzyme phenotype expressed by the transformed cells. In vitro culture was employed as a means of depleting nontransformed cells of host origin from the induced tumors. Cultured lines derived from tumors induced by each of the above agents were found to express only one of the two enzyme forms encoded by the host genotype, consistent with the probability that all the UVR-, MCA-, and BP-induced tumors examined in this study were monoclonal in origin. For further substantiation of the monoclonality of UVR-induced tumors, 2 UVR-induced tumors were enzymatically dissociated immediately following excision from the primary hosts, and the resulting cell suspensions were cloned in soft agar. Upon analysis, each set of clones selected in soft agar expressed only a single PGK-1 enzyme form. To rule out the possibility that the apparent monoclonality of culture-adapted tumor lines was due to in vitro selection, tumors that arose in UVR-treated PGK-1a/b female heterozygote mice were transplanted into PGK-1a and PGK-1b homozygous recipients. These transplanted tumors expressed a single PGK-1 allozyme following growth in recipients that were genetically homozygous for the major PGK-1 enzyme form expressed by the tumor prior to transplantation. These data strongly support the concept that most, if not all, UVR-induced tumors are derived from the progeny of a single transformed cell. This observation is important to the understanding of the nature of tumor-specific transplantation antigens expressed by individual UVR-induced tumors and indicates that such antigens also are clone specific. In addition, the results of this study indicate that polyclonality does not play a significant role in the generation of cellular and phenotypic heterogeneity known to be present within individual UVR-induced tumors.

Distribution of terminal deoxynucleotidyl transferase activity between peaks I and II in host and donor thymic cells of bone-marrow-restored mice after X-irradiation.

The terminal deoxynucleotidyl transferase (TdT) activity and its distribution between peaks I and II after chromatographic elution were studied on days 15 and 17 after X-irradiation, in host- and donor-derived thymic cells of lethally irradiated (9 Gy) mice restored with BM cells. It was found that the population derived from the surviving host thymocytes differed markedly from the donor-derived population. The cells of host origin has a low TdT activity especially in peak II and the ratio peak I/peak II remained close to 1 instead of 0.1 in controls. These alterations reflect a reduced replication rate and possibly a modification of the cellular metabolic activity (phosphorylation-dephosphorylation). In contrast, the donor-derived elements displayed a very high TdT activity related to their elevated rate of replication, and the ratio peak I/peak II which was close to 1 on day 15 returned rapidly to normal. The impaired replication ability and the metabolic alteration of the host cells might be attributed either to a specific property of the radiation-resistant thymocytes or to residual cellular injury or to a combination of both.

Sensitivity of Long-Standing Xenografts of Rat Hearts to Humoral Antibodies

Long-standing rat skin xenografts on immunosuppressed mice are known to become insensitive to destruction by mouse anti-rat serum. Our present experiments demonstrate that long-standing primarily vascularized rat-to-mouse cardiac xenografts, unlike skin, remain fully sensitive to antiserum-mediated destruction, even in mice also bearing a long-standing skin graft that is resistant to antiserum. It appears that skin grafts become resistant to antiserum-mediated destruction because of extensive replacement of the endothelium by cells of host origin. The hearts remain sensitive to antiserum since such an extensive endothelial replacement would not occur as readily in these whole organ grafts.

Serological analysis of thymus and spleen grafts

Thymus and spleen grafts from neonatal C57BL mice were implanted beneath the kidney capsule of (A x C57BL) F1 hybrids. At various intervals after implantation, the grafts were analyzed serologically. Cells of each graft were tested for the presence of cells of host origin, TL (thymus-leukemia) antigenicity, and sensitivity to the cytotoxic effect of guinea pig serum (GPS). Thymus grafts showed partial repopulation by host cells 11 days after grafting, and some grafts were completely repopulated by host cells 13 days after grafting. All thymus grafts were fully repopulated 18 days after grafting. With one exception, thymus grafts contained no significant number of TL-positive cells within 14 days after grafting. TL-positive cells appeared in thymus grafts examined 15 days after implantation, and their number increased up to the 18th day after implantation. Cells residing in thymus grafts remained sensitive to GPS throughout the period of observation. The acquisition of thymus-distinctive serological properties by host cells repopulating thymus grafts was similar in intact and in thymectomized recipients. Spleen grafts were completely repopulated by host cells as early as 8 days after grafting. The cells residing in spleen grafts remained TL-negative throughout the period of observation, and were refractory to the cytotoxic effect of GPS. It is thus apparent that, while both spleen and thymus grafts are invaded by TL-negative cells, only those entering the thymus acquire the antigen. The nature of the process by which the thymus endows thymus-distinctive properties on cells entering it is discussed.