[Experimental principles of stem cell transplantation].
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Blood Transfusion↗
Biomedical subjects
T M Fliedner
Publications and source records attributed to T M Fliedner.
[Medical management of immediately affected persons in the Chernobyl reactor accident].
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Accidents, Occupational↗
Histochemical studies on the pancreas of dogs rescued by fetal liver cell transplantation after lethal total body X-irradiation.
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Animals↗
Degeneration and atrophy of the thymus of lethally irradiated dogs, rescued by transfusion of cryopreserved autologous blood leukocytes.
Dogs exposed to a fatal radiation dose of 12 Gy were rescued by transfusion of autologous blood leukocytes. A severe acute and long-lasting damage to the thymus was observed. The acute damage, as observed on the tenth day, consisted of a marked reduction in the number of lymphocytes, degeneration of Hassall's bodies, and hemorrhage. Long-term effects, observed several months after irradiation, were partial to total atrophy of the thymus. Regeneration, when it occurred, was limited to a few small isolated areas in which lymphopoiesis was supported by epithelial reticular cells. In contrast, the lymph nodes of all dogs had abundant cortical lymphopoiesis. The abundant hemopoiesis present in the marrow from the tenth day after irradiation until the end of the observation period should have provided sufficient circulating precursor cells to seed the thymus and regenerate the organ to the same extent as that observed in the other blood-forming organs. The impairment of lymphopoietic regeneration in the thymus seems to be due, therefore, to damage caused by irradiation on the specific stroma of the organ, which is not able to support such activity.
Animals↗
The fetal liver as an alternative stem cell source for hemolymphopoietic reconstitution.
In mammalian ontogeny, the liver constitutes the primary hematopoietic organ for some time. Fetal liver cells (FLC) are rich in hematopoietic stem cells with a high proliferative potential but contain few post-thymic T cells. In animal studies, FLC restored hematopoiesis without severe graft-versus-host disease. However, genetic disparity between donor and host frequently limited durable engraftment and prevented or protracted complete immune reconstitution in most fully allogeneic recipients. Some children with severe combined immunodeficiency have been cured by FLC infusion, whereas favorable effects in aplastic anemia, acute leukemia, and inborn errors of metabolism have been limited and badly understood. Fetal liver transplantation in animals may serve as a model for the analysis and management of complications associated with the transfer of purified hematopoietic stem cell grafts and aid in the development of future therapeutic strategies requiring rapidly proliferating stem cell populations.
Anemia, Aplastic↗
Acute and long-term alterations in the granulocyte/macrophage progenitor cell (GM-CFC) compartment of dogs after partial-body irradiation: irradiation of the upper body with a single myeloablative dose.
The acute and long-term effects of a single dose of partial-body irradiation on the granulocyte/macrophage progenitor cell compartment were studied in dogs. A myeloablative dose of 11.7 Gy (dose rate 6.5 cGy/min) was given to the upper body which contains approximately 70% of the total bone marrow mass. The lower part of the body (pelvis, lower extremities and tail) was shielded by a lead box. In the non-irradiated bone marrow, the concentration of the GM-CFC/10(5) mononuclear cells was slightly decreased within the first 7 days and showed some fluctuations around the normal value for several weeks thereafter. In the irradiated bone marrow, virtually no GM-CFC could be detected on day 1 after exposure. Beginning on day 7, a continuous increase took place up to day 21 when the GM-CFC concentration reached between 25% (sternum) and 43% (humerus) of the initial value. No further increase took place up to day 80. Between day 120 and 380 a secondary increase was observed which reached near-normal bone marrow GM-CFC concentrations. The blood GM-CFC concentration first showed a strong depression followed by a transient increase between day 10 and 30. This coincided with GM-CFC normalization in the protected bone marrow as well as with the initial phase of regeneration in the irradiated sites. A prolonged secondary long-lasting depression between day 33 and 120 amounted to 20 to 50% of normal values. This depression was closely related to the stagnation in the GM-CFC recovery in the irradiated bone marrow sites. The GM-CFC concentration in the blood was found to be supranormal at day 380 when the bone marrow GM-CFC had recovered. The colony stimulating activity in the serum showed an increase within the first 20 days after exposure, that is, within the same interval the bone marrow GM-CFC concentration experienced the strongest alterations, and was inversely related to the changes in the blood granulocyte values.
Animals↗
The development of radiation late effects to the bone marrow after single and chronic exposure.
The marrow is a tissue distributed in numerous skeletal parts and works as an organ which is composed of a haemopoietic cell parenchyma and a supporting stroma. The pathophysiological mechanisms involved in the radiation-induced late effects depend mainly on the damage produced to each of these elements. Parenchymal cell damage ends with a failure of the stem cell pool to supply an adequate number of highly differentiated functional blood cells and is clinically manifested as aplastic anaemia or leukaemia. The effects of radiation on the haemopoietic stem cell can be measured by means of spleen colony forming units (CFU-S) in rodents. The self-maintaining capacity of the CFU-S was found to be lower than normal 16 weeks after a dose of 0.64 Gy. In larger animals it is only possible to measure the activity of some of the progenitor cells, estimating the number of granulocyte-macrophage colonies in culture (CFU-GM) as an indicator of stem cell changes. Their number in the blood is about 50 per cent of normal even 160 days after about 0.78 Gy. The stromal cells are also radiosensitive if measured with respect to their capacity to support long-term cell replication in vitro. Marrow fibrosis develops after single, repeated and chronic radiation exposure, and a dose of 40 Gy impairs the capacity of the marrow to support haemopoiesis.
Anemia, Aplastic↗
Autologous transplantation of blood-derived hemopoietic stem cells after myeloablative therapy in a patient with Burkitt's lymphoma.
A patient with Burkitt's lymphoma in complete remission received myeloablative consolidation treatment with superfractionated total body irradation (1,320 rad) and cyclophosphamide (200 mg/kg) followed by autologous transplantation of previously harvested and cryopreserved blood-derived hemopoietic stem cells. Seven successive leukaphereses were performed to yield a total of 55.2 X 10(9) mononuclear cells (MNC) comprising 15.1 X 10(6) CFU-GM or 4.34 X 10(6) CFU-GEMM. Following autologous blood stem cell transplantation (ABSCT), reconstitution of all cell lines occurred very rapidly, ie, 1,000 leucocytes per microL were reached after nine days, 500 granulocytes and 50,000 platelets per microL after ten days. B cells reached normal values around day 35 post-transplantation. CFU-GM first appeared in the circulating blood exhibiting an enormous overshoot. Some days later CFU-GM also appeared in the marrow. The kinetics and pattern of hemopoietic reconstitution after myeloablative treatment and ABSCT provide clear evidence that blood-derived hemopoietic stem cells are capable of completely restoring hemopoietic function in man. A possible reconstitutive advantage of blood over marrow-derived stem cells is discussed.
Adult↗
Histochemical studies on the effects of lethal total body X-irradiation on the pancreas of dogs rescued by autologous bone marrow transplantation.
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Animals↗
CFU-F from dog marrow: a colony assay and its significance.
A colony assay method is described for studying dog fibroblast colony development in marrow cells derived from resected ribs. The assay showed an increased number of fibroblast colony forming units (CFU-F) in cell suspensions prepared from resected ribs compared to cell suspensions prepared from bone marrow aspirates or from peripheral blood. A linear relationship between the number of cells plated and the number of fibroblastoid colonies was demonstrated in each case. The proportion of phagocytic cells was lower in cultures prepared from resected ribs than in those prepared from bone marrow aspirates. Staining for acid phosphatase and with sudan black showed differences between phagocytic cells and non-phagocytic fibroblasts. When left in plastic dishes for 2 hrs, 81% +/- 10% of the CFU-F adhered to the plastic dishes. Velocity sedimentation separation showed a modal sedimentation rate of 6.49 mm/h.
Animals↗
Repopulating potential of canine bone marrow cells: differences between large and small cells separated by velocity sedimentation.
This study compares the pattern of haemopoietic recovery in dogs after total-body irradiation and transfusion of different populations of cryopreserved autologous bone marrow cells. Dogs in group 1 received unseparated marrow cells. Group-II dogs were transfused with small (less than 5.1 mm/h) and group-III dogs with large (greater than 7.1 mm/h) bone marrow cells, separated by velocity sedimentation. Myeloid progenitor cells (CFU-GM) present in slowly sedimenting cell fractions were characterized by a higher radiosensitivity in vitro and a lower proportion of cells in S-phase as compared to rapidly sedimenting CFU-GM. Although autografts in all groups contained comparable numbers of CFU-GM, transfusions resulted in different patterns of recovery. Fractions of small bone marrow cells contained most of the pluripotent stem cells, leading to speedy haematological reconstitution and long-term survival. The pattern of early recovery was similar in dogs of group I and of group II. In group III, the recovery in all cell lineages was delayed, going along with marked extramedullary haemopoiesis. The data may indicate the limits of committed progenitors in reflecting pluripotential stem cells, in particular, if bone marrow grafts have been modified in vitro. Differences in the repopulating potential of the graft might be reflected by distinct physical and biological properties of the CFU-GM.
Animals↗
Bone marrow structure and its possible significance for hematopoietic cell renewal.
The authors review the progress made during the last quarter of a century in the fields of hematopoietic cellular proliferation and differentiation in relation to the bone marrow structure and the microenvironment provided by the marrow stroma in which unlimited self-renewal occurs. The marrow is conceived of as an organ in which the stroma originates from local mesenchymal elements which form a vascularized and innervated matrix, seeded later by blood-borne stem cells. Transplantation studies using total-body-irradiated dogs show that stem cells derived from the marrow, as well as those from the blood and from the fetal liver, are able to repopulate a marrow rendered aplastic by irradiation. By grafting equal numbers of GM-CFU from peripheral blood and bone marrow, a faster hemopoietic reconstitution is provided by blood-derived stem cells. The most efficient stem cells in the long range are those derived from fetal liver. Bone marrow and peripheral blood GM-CFU differ in some in vitro characteristics such as radiation sensitivity. These peripheral blood cells are more radiosensitive than those derived from the marrow. Autografting of bone marrow mononuclear cell fractions obtained by velocity sedimentation techniques demonstrates that the fraction of small mononuclear cells holds a repopulating potential similar to that of circulating blood stem cells. The cells collected in fraction 2 of a discontinuous albumin gradient contain most of the blood stem cells and repopulate the marrow without causing GVHD, while cells collected in fractions 3 and 4 contain a minimal amount of stem cells and cause severe GVHD.
Animals↗
Fetal liver transplantation in the dog. II. Repopulation of the granulocyte-macrophage progenitor cell compartment by fetal liver cells from DLA-identical siblings.
The restoration of the granulocyte-macrophage progenitor cell (CFU-GM) compartments in blood and bone marrow, and the recovery of blood monocytes were followed for up to one year in ten beagles that had been exposed to fractionated (3 X 6 Gy) total-body irradiation before being transfused with cryopreserved fetal liver cells (FLC) from sibling donors that were genotypically matched for dog leukocyte antigens. Grafts contained 0.2-1.6 X 10(8) mononuclear cells and 0.9-19.8 X 10(4) CFU-GM/kg body weight. Numbers of circulating monocytes rose parallel to granulocyte numbers after day 6 and became normal by day 18 posttransplant. In bone marrow aspirates, low numbers of CFU-GM were detected on day 3 and their incidence per 10(5) mononuclear cells was normal after day 14. Circulating CFU-GM were present in significant numbers by day 7 and their elevated concentration per milliliter of blood after day 14 continued for one year. Dextran sulfate injection mobilized normal numbers of CFU-GM into the blood early after transplantation, and spontaneously circulating CFU-GM in a later phase did not differ from blood progenitors of normal animals with respect to radiation sensitivity and sedimentation velocity. Thus, FLC transplantation effected a rapid restoration of granulopoiesis and monocytopoiesis, which was reflected at both the level of mature blood cells and the compartments of CFU-GM in blood and bone marrow, underlining the high repopulating capacity of fetal liver stem cells.
Animals↗
Allogeneic transplantation of blood stem cells concentrated by density gradients.
Discontinuous albumin density gradients were used to physically separate hemopoietic cells from immunocompetent lymphocytes in peripheral blood mononuclear cells of dogs. Transplantation of these stem cell concentrates into lethally irradiated allogeneic recipients restored hemopoiesis in nine of 12 animals. Tolerance and long-term survival, however, were achieved only in pairs matched for major histocompatibility antigens. These animals showed stable chimerism in bone marrow cells but transiently regenerated autochthonous cells demonstrable only in the peripheral blood. As compared with earlier studies using grafts of unseparated peripheral blood mononuclear cells, the numbers of CFUc transplanted were similar; the number of mononuclear cells, however, was reduced by a factor of 60. It thus appears that the granulocyte-macrophage colony-forming assay (GM-CFUc) is able to predict within limits the hemopoietic potential of a graft. Although very low numbers of blood-derived CFUc were effective (33,000-42,000/kg body weight), the superiority of blood stem cells over bone marrow grafts remains to be established.
Animals↗
Gradient separation of granulocytic progenitor cells (CFUc) from human blood mononuclear leukocytes.
Two different density-gradient techniques were compared for separation of human blood-derived granulocyte-macrophage progenitor cells (CFUc) from immunocompetent cells using albumin or Percoll as gradient media. Mean CFUc enrichment by means of discontinuous albumin gradient was twofold, whereas Percoll gradients yielded about 20- to 40-fold enrichment of CFUc in relation to lymphocytes. The pH of gradient media proved to have a major influence on separation quality. Based on these results and the development of a new density-separation technique in a 600-ml plastic bag, replacing customary Ficoll-Hypaque gradients, a method for large-scale purification of CFUc from leukapheresis-derived leukocytes has been constructed, working in a closed, sterile system. About 11 X 10(9) mononuclear cells containing 6 X 10(5) CFUc could be processed within 10 h. Lymphocytes were reduced to less than 1% of their initial number, and an average of about 50% of the original CFUc could be recovered.
Cell Separation↗
Fetal liver transplantation in the dog. I. Restoration of hemopoiesis with cryopreserved fetal liver cells from DLA-identical siblings.
Fetal liver cells (FLC) were obtained from beagle fetuses 52 days postconception, and were cryopreserved prior to transplantation into ten sibling recipients that had previously been exposed to total-body irradiation delivered in 3 fractions of 6 Gy each at 4 days, 2 days, and 2 hr before grafting. Donors and hosts were genotypically identical for dog leukocyte antigens (DLA)-A, B, and D. A rapid and lasting engraftment was achieved in all animals following the transfer of 0.2 X 10(8) to 1.6 X 10(8) mononuclear FLC/kg body weight, which were equivalent to 0.9 X 10(4) to 19.8 X 10(4) granulocyte/macrophage progenitor cells (CFU-GM)/kg. Between days 14 and 20 posttransplant pretreatment levels were detected for blood granulocytes, between days 23 and 28 for circulating platelets, and between days 35 and 40 for the erythrocyte count and hemoglobin concentration. Increasing the number of CFU-GM transfused resulted in an accelerated granulocyte and platelet recovery. Bone marrow cells were of donor origin throughout the observation interval, but declining proportions of host lymphocytes circulated in the peripheral blood during the initial recovery phase. In two dogs, skin alterations that might indicate slight graft-versus-host disease (GVHD) were noted following days 20 and 70, respectively. Six recipients had to be sacrificed due to inanition, probably secondary to radiation-induced pancreatic insufficiency two to three months after grafting. The results of this study indicate that cryopreserved FLC are highly effective in restoring hemopoiesis in DLA-compatible sibling dogs. Transplantation of canine FLC may prove valuable in analyzing mechanisms pathogenetically related to graft rejection or to the development of GVHD following the transfer of T-cell-depleted hemopoietic grafts at a preclinical stage.
Animals↗
Recovery of immune functions in dogs after total body irradiation and transplantation of autologous blood or bone marrow cells.
The restoration of immune functions was followed in dogs for 101 days after fractionated total body irradiation and autologous transfusion of peripheral blood leukocytes (PBL) or bone marrow (BM) cells. Median numbers of 0.9 X 10(5) granulocyte-macrophage progenitor cells per kilogram of body weight were transferred in either group of recipients. The following parameters recovered more rapidly in PBL recipients as opposed to BM recipients: total blood lymphocyte, T- and B-cell counts, serum levels of immunoglobulins IgM and IgA, in vitro blastogenic responses after stimulation with concanavalin A and pokeweed mitogen, and in vitro plasma cell formation after polyclonal B-cell activation with pokeweed mitogen with or without lipopolysaccharide. No major differences were noted for the restoration of serum IgG levels. Circulating lymphocyte and T-cell numbers remained subnormal for more than three months in both groups, whereas B-cell numbers and serum levels of IgA continued to be depressed in BM recipients only. Thus, autologous PBL restored immune functions more rapidly than did BM. Transplantation of PBL, alone or in addition to autologous BM, might also shorten the period of immunodeficiency after cytoreduction in a variety of malignancies in man.
Animals↗
Hemopoiesis and immune functions in dogs following fetal liver transplantation.
Ten beagles were exposed to total body X-irradiation (3 X 6 Gy) and rescued with cryopreserved fetal liver cells from DLA-identical siblings obtained around the 52nd day of gestation. Grafts contained 0.2-1.6 X 10(8) mononuclear cells/kg and 0.9-19.8 X 10(4) granulocyte-macrophage progenitor cells/kg. Hemopoiesis and immune functions were followed for up to one year after fetal liver transplantation (FLT). There was a prompt engraftment in all recipients. Bone marrow metaphases were always of donor origin, whereas some host lymphocytes circulated for 2-3 months. Blood granulocytes and monocytes rose to pre-treatment levels within 2-3 weeks of FLT and platelets and erythrocytes were normal within 3-4 and 5-6 weeks, respectively. The relative incidence of bone marrow CFU-GM was normal by day 14 and the absolute numbers of circulating CFU-GM remained elevated for one year after day 14. Blood lymphocytes reached control numbers between days 35 and 101 with a faster B cell than T cell recovery. Their response to mitogen stimulation was normal by day 75, while the mixed lymphocyte reaction tended to be reduced for one year. Serum levels of IgM (day 35) and IgG (day 49) recovered earlier than IgA levels (day 270). Thus, cryopreserved canine fetal liver cells can restore hemopoiesis and immunocompetence with considerable rapidity in histocompatible, adult siblings pre-treated with total body irradiation, and, since they lack mature T cells, may be used to analyze effector mechanisms that mediate rejection of T cell-depleted allografts under less favourable conditions.
Animals↗