骨髓基质细胞的辐射效应及其临床意义

小鼠骨髓基质细胞团在γ线照射后的Do值为2.40Gy,但其成灶能力损伤后持续时间较久。正常骨髓基质细胞能促进骨髓GM-CFU-C的生长;照射10-80Gy后的骨髓基质细胞失去这种促进作用。文中讨论了骨髓基质细胞的辐射效应及其临床意义,提出了谨慎选择放射治疗剂量的必要性。     

Radiosensitivity of stromal precursors and mature hematopoietic stromal cells in a culture

Radiosensitivity of hemopoietic stroma precursors from a long-term culture of murine bone marrow, as measured by the adherent cell layer implantation techniques, was characterized by D0 = 3.02 +/- 0.7 Gy and n = 1.6. Mature cells of the hemopoietic microenvironment survived after doses of up to 100 Gy. Their irreversible damage was only observed after 150-200 Gy irradiation. The results obtained support the suggestion of different histogenetical origin of the hemopoietic and stromal precursors.     

2 populations of bone marrow cells transfering the hematopoietic environment]

A bone marrow fragment transplanted under the kidney capsule created a focus of ectopic hemopoiesis, whose isze, measured by the number of hemopoietic cells, was proportional to the implant size. Dimensions of the focus proved to be 11/2--21/2 greater in the irradiated than in the intact recipients. Cells building up the focus of heterotopic hemopoiesis had a different radiosensitivity in the intact and irradiated recipients--their Do constituted about 160 and 350 rad, respectively. In this connection it is supposed that two cell populations of precursors took part in the creation of the focus. Their possible relations with the determined and inducible osteogenic precursor cells are discussed.     

Survival of human normal thyroid cells after X-ray irradiation

Normal thyroid cells from 25 individuals treated surgically for malignant or benign thyroid tumour were cultured in vitro and radiation induced cytotoxicity was studied. The mean lethal dose (Do), quasi-threshold (Dq), and extrapolation number (n) of survival curves of actively dividing thyroid cells assayed by colony formation were estimated to be 92.9 +/- 2.8 cGy (rad), 58.1 +/- 6.9 cGy and 2.0 +/- 0.1, respectively (average for 25 individuals +/- standard error). These results suggest that proliferating human thyroid cells are more sensitive to X-rays than most other nonhaematologic mammalian cells in similar assays. Cell survival was not significantly affected by sex, age, disease or exposure to atomic bomb radiation of the cell donor. However, the number of samples currently available is too small for definite conclusions in this regard.     

Population Dynamics of Clonogenic Stromal Precursor Cells in Hemopoietic and Lymphoid Organs during Bone Marrow Regeneration

This paper describes our study on the regeneration of hemopoietic and stromal components of bone marrow after mechanically emptying the medullar cavity of the guinea pig tibia. The intensity of hemopoiesis was determined from the number of hemopoietic cells, while the concentration and total number of stromal precursor cells were used to estimate the ability of the bone marrow to produce stromal structures, including its ability to restore a specific microenvironment. We found that there was no direct correlation between the recovery characteristics of hemopoietic and stromal cells. An increase in the population size of stromal precursor cells takes place early after curettage, and stromal fibroblasts become phosphatase-positive according to Gomori, which is characteristic of osteogenic tissue. We have also demonstrated that curettage of 3–5 tubular bones results in the growth of this cell population in the bone marrow of nonoperated bones and even in the spleen, which in guinea pigs participates only in lymphopoiesis.     

Osteogenic stem cells of the bone marrow

This paper presents literature and author's own data demonstrating that bone marrow contains determined osteogenic precursor cells with high potential to differentiation. They are stem cells of the bone and belong to the stromal cell line of the bone marrow which is histogenetically independent of hemopoietic cells. The paper presents detailed analysis of bone marrow stromal cells (CFUf) as well as of their osteogenic properties and requirements in growth factors. In conclusion mutual growth-stimulating interactions in the system of hemopoietic stromal cells are reviewed.     

Pre CFU-F in bone marrow cultures from children with hematopoietic disease including acute leukemia

Stromal precursor cells from bone marrow aspirates of children have been studied in culture. In 7 day liquid cultures normal individuals and patients with acute leukemia in remission grew 110 +/- 50 CFU-F and 100 +/- 40 CFU-F (colony forming unit--fibroblasts) respectively, per 6 X 10(5) buffy coat mononuclear cells. Staining with monoclonal antibodies suggests that stromal cells from CFU-F colonies are fibroblasts. CFU-F colony growth from the bone marrow of patients with active leukemia was low. After cultivation periods of more than 21 days, we observed, in addition, still more immature, clonogenic fibroblast precursor cells, "pre CFU-F", and round cells attached to stromal cells from pre CFU-F colonies. From the round cells, we have passaged pre CFU-F and CFU-GM (colony forming unit--granulocytic, monocytic) in secondary cultures. Our observations are in agreement with the concept that the bone marrow stromal cell matrix serves as a sanctuary for reversibly attached clonogenic cells of both the hematopoietic and fibroblast lineages.     

Expression of bone morphogenetic proteins in stromal cells from human bone marrow long-term culture.

Highly purified primitive hemopoietic stem cells express BMP receptors but do not synthesize bone morphogenetic proteins (BMPs). However, exogenously added BMPs regulate their proliferation, differentiation, and survival. To further explore the mechanism by which BMPs might be involved in hemopoietic differentiation, we tested whether stromal cells from long-term culture (LTC) of normal human bone marrow produce BMPs, BMP receptors, and SMAD signaling molecules. Stromal cells were immunohistochemically characterized by the presence of lyzozyme, CD 31, factor VIII, CD 68, S100, alkaline phosphatase, and vimentin. Gene expression was analyzed by RT-PCR and the presence of BMP protein was confirmed by immunohistochemistry (IHC). The supportive role of the stromal cell layer in hemopoiesis in vitro was confirmed by a colony assay of clonogenic progenitors. Bone marrow stromal cells express mRNA and protein for BMP-3, -4, and -7 but not for BMP-2, -5, and -6 from the first to the eighth week of culture. Furthermore, stromal cells express the BMP type I receptors, activin-like kinase-3 (ALK-3), ALK-6, and the downstream transducers SMAD-1, -4, and -5. Thus, human bone marrow stromal cells synthesize BMPs, which might exert their effects on hemopoietic stem cells in a paracrine manner through specific BMP receptors.     

The effect of low dose rate on recovery of hemopoietic and stromal progenitor cells in gamma-irradiated mouse bone marrow

Long-term recovery of mouse hemopoietic stem cells (CFU-S and CFU-S per colony), granulocyte-macrophage precursor cells (GM-CFC), and stromal colony-forming units (CFU-F) after doses up to 12.5 Gy was almost complete by 1 year when the dose rate was reduced to 0.0005 Gy/min compared to incomplete recovery after doses up to only 6.5 Gy given at greater than 0.7 Gy/min. This sparing effect of dose rate on long-term hemopoietic recovery is in contrast to the generally reported lack of dependence on dose rate for acute survival of hemopoietic progenitors after doses up to 5 Gy. The present results are compatible with the hypothesis that good recovery of the stroma should be reflected in the long-term recovery of hemopoiesis.     

Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.     

The effects of graded doses of 1 MeV fission neutrons or X rays on the murine hematopoietic stroma.

The acute radiosensitivity in vivo of the murine hematopoietic stroma for 1 MeV fission neutrons or 300 kVp X rays was determined. Two different assays were used: (1) an in vitro clonogenic assay for fibroblast precursor cells (CFU-F) and (2) subcutaneous grafting of femora or spleens. The number of stem cells (CFU-S) or precursor cells (CFU-C), which repopulated the subcutaneous implants, was used to measure the ability of the stroma to support hemopoiesis. The CFU-F were the most radiosensitive, and the survival curves after neutron and X irradiation were characterized by D0 values of 0.75 and 2.45 Gy, respectively. For regeneration of CFU-S and CFU-C in subcutaneously implanted femora, D0 values of 0.92 and 0.84 Gy after neutron irradiation and 2.78 and 2.61 Gy after X irradiation were found. The regeneration of CFU-S and CFU-C in subcutaneously implanted spleens was highly radioresistant as evidenced by D0 values of 2.29 and 1.49 Gy for survival curves obtained after neutron irradiation, and D0 values of 6.34 and 4.85 Gy after X irradiation. The fission-neutron RBE for all the cell populations was close to 3 and varied from 2.77 to 3.28. The higher RBE values observed for stromal cells, compared to the RBE of 2.1 reported previously for hemopoietic stem cells, indicate that stromal cells are relatively more sensitive than hemopoietic cells to neutron irradiation.     

Ultrastructural characteristics of stromal mechanocytes and their interaction with hematopoietic cells in regenerating bone marrow transplants

Extramedullar grafts of the bone marrow have been studied electron microscopically 2-8 days after transplantation. The data on ultrastructural peculiarities of the stromal mechanocytes have been obtained at various stages of their differentiation, as well as topographic interrelationships between the mechanocytes and the hemopoietic cells. Digital junctions are described between the stromal mechanocytes (primitive) and the hemopoietic cells (in 3-day-old grafts) which are, probably, the first morphological signs demonstrating restoration of the hemopoietic microenvironment in the bone marrow grafts.     

Effect of Continuous γ-Irradiation at Low Doses on Clonogenic Hemopoietic (CFU-S) and Stromal (CFU-F) Cells of Bone Marrow

We studied the effects of low doses of continuous -irradiation (Co⁶⁰, 10 days, mean daily dose power 1.5-2.0 mGy, total dose 15 mGy) on hemopoietic and stromal progenitor cells of murine bone marrow. The content of hemopoietic clonogenic cells representing a younger (CFU-S-11) and more mature (CFU-S-7) categories in the compartment of stem cells was determined in the bone marrow. The state of bone marrow stroma was estimated by the method of in vitro cloning according to the number of progenitor cells that form colonies of fibroblasts (CFU-F) and by the method of ectopic transplantation according to the capacity of stroma of organizing and building new hemopoietic territories. Continuous -irradiation at low doses, that were by one order of magnitude lower than those inducing hermesis, exerted a stimulating effect on both hemopoietic (CFU-S) and stromal (CFU-F) progenitor cells. The number of CFU-S in the compartment of stem cells of the bone marrow markedly increased and they formed larger hemopoietic territories but these cells appeared to create a qualitatively different microenvironment, which stimulated the proliferation of CFU-S.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

Hepatocyte growth factor induces proliferation and differentiation of multipotent and erythroid hemopoietic progenitors

Hepatocyte growth factor (HGF) is a mesenchymal derived growth factor known to induce proliferation and "scattering" of epithelial and endothelial cells. Its receptor is the tyrosine kinase encoded by the c- MET protooncogene. Here we show that highly purified recombinant HGF stimulates hemopoietic progenitors to form colonies in vitro. In the presence of erythropoietin, picomolar concentrations of HGF induced the formation of erythroid burst-forming unit colonies from CD34-positive cells purified from human bone marrow, peripheral blood, or umbilical cord blood. The growth stimulatory activity was restricted to the erythroid lineage. HGF also stimulated the formation of multipotent CFU- GEMM colonies. This effect is synergized by stem cell factor, the ligand of the tyrosine kinase receptor encoded by the c-KIT protooncogene, which is active on early hemopoietic progenitors. By flow cytometry analysis, the receptor for HGF was found to be expressed on the cell surface in a fraction of CD34+ progenitors. Moreover, in situ hybridization experiments showed that HGF receptor mRNA is highly expressed in embryonic erythroid cells (megaloblasts). HGF mRNA was also found to be produced in the embryonal liver. These data show that HGF plays a direct role in the control of proliferation and differentiation of erythroid progenitors, and they suggest that it may be one of the long-sought mediators of paracrine interactions between stromal and hemopoietic cells within the hemopoietic microenvironment.     

IL-17 receptor knockout mice have enhanced myelotoxicity and impaired hemopoietic recovery following gamma irradiation

IL-17A is a T cell-derived proinflammatory cytokine required for microbial host defense. In vivo expression profoundly stimulates granulopoiesis. At baseline, the hemopoietic system of IL-17R knockout mice (IL-17Ra(-/-)) is, with the exception of increased splenic progenitor numbers, indistinguishable from normal control mice. However, when challenged with gamma irradiation, hemopoietic toxicity is significantly more pronounced in IL-17Ra(-/-) animals, with the gamma irradiation-associated LD(50) being reduced by 150 rad. In spleen-derived T cells, gamma irradiation induces significant murine IL-17A expression in vivo but not in vitro. After sublethal radiation injury (500 rad), the infusion of purified CD4(+) T cells enhances hemopoietic recovery. This recovery is significantly impaired in IL-17Ra(-/-) animals or after in vivo blockade of IL-17Ra in normal mice, resulting in a reduction of hemopoietic precursors by 50% and of neutrophils by 43%. Following sublethal radiation-induced myelosuppression, in vivo overexpression of murine IL-17A in normal mice substantially enhanced granulopoietic restoration in mice with a 4-fold increase in neutrophils and splenic precursors on day 8 (CFU-granulocyte-macrophage/granulocyte-erythrocyte-megakaryocyte-monocyte, CFU-high proliferative potential), as well as 2- and 3-fold increases of bone marrow precursors, respectively. This establishes IL-17A as a hemopoietic response cytokine to radiation injury in mice and an inducible mechanism that is required for recovery of granulopoiesis after radiation injury.     

Characteristics of hematopoiesis regeneration in subcutaneous bone implants in aging mice

Femurs from both young and old mice were implanted subcutaneously to young syngeneic recipients. 2-2.5 months after surgery femoral marrow grafts were repopulated with nucleated cells and macrophage-granulocyte precursor cells irrespective of the donor's age. Marrow depletion of femurs prior to implantation caused a sharp decrease in hemopoietic repopulation of implants from old, but not young, donors. It is concluded that age-dependent functional peculiarities of stromal microenvironment can be visualized owing to the extraordinary local regenerative demand.     

A stromal cell line from rainbow trout spleen, RTS34st, that supports the growth of rainbow trout macrophages and produces conditioned medium with mitogenic effects of leukocytes

Summary A rainbow trout spleen cell line, RTS34, was developed from a long-term hemopoietic culture. This cell line consisted of a mixed stromal cell layer with an associated cell population of macrophage-like cells that formed proliferative foci and released nonadherent progeny cells into the culture medium. A stromal cell line, RTS34st, was isolated from the RTS34 cell line. RTS34st cultures contained cells with fibroblast-like and epithelial-like morphologies and showed enhanced [³H]thymidine incorporation in response to either FBS or rainbow trout serum. The combination of FBS and trout serum was synergistic. Conditioned medium from RTS34st stimulated thymidine incorporation by peripheral blood and head kidney leukocytes, but not by leukocytes from the spleen. In addition, RTS34st provided a hemopoietic inductive microenvironment for immature precursor cells, selectively supporting the growth of macrophage-like cells. Therefore, RTS34st appears useful for studying the different roles of the stroma in regulating hemopoiesis in fish.     

Generation of osteoclasts from hemopoietic cells and a multipotential cell line in vitro

Osteoclasts are the cells that resorb bone. It is generally presumed, on the basis of indirect experiments, that they are derived from the hemopoietic stem cell. However, this origin has never been established. We have developed an assay for osteoclastic differentiation in which bone marrow cells are incubated in liquid culture on slices of cortical bone. The bone slices are inspected in the scanning electron microscope after incubation for the presence of excavations, which are characteristic of osteoclastic activity. We have now incubated bone marrow cells at low density, or a factor-dependent mouse hemopoietic cell line (FDCP-mix A4) with 1,25 dihydroxyvitamin D3 (a hormone which we have previously found induces osteoclastic differentiation) with and without murine bone marrow stromal cells, or with and without 3T3 cells, on bone slices. Neither the bone marrow cells nor the bone marrow stromal cells alone developed osteoclastic function even in the presence of 1,25 dihydroxyvitamin D3. However, extensive excavation of the bone surface was observed, only in the presence of 1,25 dihydroxyvitamin D3, on bone slices on which bone marrow stromal cells were cocultured with low-density bone marrow cells or the hemopoietic cell line. Similar results were obtained when the bone marrow stromal cells were killed by glutaraldehyde fixation; 3T3 cells were unable to substitute for stromal cells. These results are strong evidence that osteoclasts derive from the hemopoietic stem cell and suggest that although mature osteoclasts possess neither receptors for nor responsiveness to 1,25 dihydroxyvitamin D3, the hormone induces osteoclastic function through a direct effect on hemopoietic cells rather than through some accessory cell in the bone marrow stroma. The failure of 3T3 cells, which enable differentiation of other hemopoietic progeny from this cell line, to induce osteoclastic differentiation suggests that bone marrow stroma possesses additional characteristics distinct from those that induce differentiation of other hemopoietic cells that are specifically required for osteoclastic differentiation.     

Cell lineage segregation during bursa of Fabricius ontogeny

The population dynamics of myeloid and lymphoid lineages during bursa of Fabricius ontogeny were analyzed by immunofluorescence by using two monoclonal antibodies (mAb). CL-1 mAb reacts with all chicken hemopoietic cells, except mature erythrocytes. L22 mAb reacts with bursa and bursa-derived lymphocytes, with a minor subset of macrophages and with some cells of the thymic medulla. The staining of embryonic bursas by these antibodies helps to distinguish between two different lineages of hemopoietic cells: CL-1+/L22+ cells represent B lymphocytes and a minor subset of macrophages, while CL-1+/L22- cells correspond to most of the macrophages and to the granulocytes, which disappear at the end of the embryonic life. CL-1+/L22- as well as CL-1+/L22+ cells were first observed outside the bursal rudiment. This indicates that there is a pre-bursal segregation between these two hemopoietic lineages and that two different kinds of precursors colonize the bursal rudiment at about the same time (day 9 for CL-1+/L22- cells and days 9 or 10 for CL-1+/L22+ cells). Moreover our data show that the colonization of the bursal epithelium by hemopoietic precursors is a two-step phenomenon. The first cells which enter belong to the CL-1+/L22- lineage, express Ia-like antigens at a high level, are dendritic in morphology, and represent cells of the macrophage/dendritic cell lineage. They are responsible for the formation of the epithelial bud which are then colonized by a small number of lymphoid precursors which belong to the CL-1+/L22+ lineage. Quail-chick bursa grafting experiments were also performed and the grafts were examined for CL-1 (restricted to chicken hemopoietic cells) and L22 reactivity. These observations confirmed our previous findings about the kinetics of the colonization of bursal rudiment by hemopoietic precursors and give support for a pre-bursal segregation between two hemopoietic pathways.