Hematopoietic growth factors released by marrow stromal cells from patients with aplastic anemia.

We studied the production of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6) by stromal cells from 33 patients with aplastic anemia (AA). Complete, confluent stromal layers were produced by 29 of the 33 samples using the long-term bone marrow culture (LTBMC) system. The concentration of G-CSF, GM-CSF, and IL-6 in culture media with or without interleukin-1 (IL-1) stimulation was determined by an enzyme-linked immunoadsorbent assay (ELISA). The spontaneous production of G-CSF, GM-CSF, and IL-6 did not differ significantly between normal controls and the patients with AA. The ability of stromal cells to release the three hematopoietic growth factors in response to IL-1 was either normal or elevated in all but one patient. We also studied the change in production of G-CSF, GM-CSF, and IL-6 by stromal cells before and after antilymphocyte globulin (ALG) therapy in 16 patients with AA. There was no correlation between the change in production of these cytokines and the response to ALG. In contrast to previous studies that showed a defect in the production of hematopoietic growth factors by stromal cells from patients with AA, the results indicated a normal or elevated production of G-CSF, GM-CSF, and IL-6 by marrow stromal cells in patients with AA.     

Identification of the hematopoietic growth factors elaborated by bone marrow stromal cells using antibody neutralization analysis

These studies were undertaken to characterize the subclasses of hematopoietic growth factors produced by stromal cells in long-term murine bone marrow cultures. Exposure of these cultures to extremely high doses of irradiation (500 Gy), followed by endotoxin stimulation, permitted detection and characterization of various growth factor activities in the unconcentrated conditioned medium. To determine the nature of these activities, neutralization studies were performed using antisera against the following subclasses of purified colony-stimulating factors (CSFs): purified L-cell CSF-1, recombinant granulocyte-macrophage CSF (rGM-CSF), and recombinant interleukin 3 (rIL3). The antiserum against CSF-1 consistently abrogated 100% of the CSF bioactivity in irradiated stromal cell-conditioned medium (CM) but was only capable of neutralizing 62%-91% of the bioactivity in endotoxin-stimulated, irradiated stromal cell-CM. Antisera against rGM-CSF and rIL3 demonstrated variable effects. When the antisera were used in combinations, only the mixture of anti-CSF-1 + anti-GM-CSF resulted in 100% neutralization of the activities in endotoxin-stimulated, irradiated stromal cell-CM. This CM stimulated the IL3/GM-CSF-responsive cell line FDC-P1 but not the IL3-responsive (GM-CSF-unresponsive) cell line 32D cl-23. The FDC-P1 growth-promoting activity was inhibited only by the antiserum against GM-CSF and not by antiserum against IL3. These experiments indicate that stromal cells from long-term bone marrow cultures can produce and release CSF-1 and GM-CSF while the production of IL3 in this system, if there is any, could not be demonstrated.     

Monocytes stimulate fibroblastoid bone marrow stromal cells to produce multilineage hematopoietic growth factors

In previous studies we have found that monocytes produce soluble factors that stimulate human umbilical vein endothelial cells to produce granulocyte-macrophage colony-stimulating activity (CSA), burst- promoting activity (BPA), and megakaryocyte colony-stimulating activity (Meg-CSA) as well as factors that stimulate T lymphocytes and neonatal fibroblasts to produce CSA. To test the hypothesis that monocytes would similarly stimulate the production of hematopoietic growth factors by autologous bone marrow stromal cells, multiply-passaged adherent fibroblastoid cells derived from the bone marrow of normal volunteers were exposed to conditioned media prepared by incubating autologous peripheral blood monocytes in complete medium for three days. When conditioned media from stromal cells incubated in monocyte-conditioned medium were compared with those of stromal cells cultured in the absence of monocyte-conditioned medium, BPA was increased fourfold and CSA was increased more than 30-fold. We conclude that mononuclear phagocytes recruit stromal cells of the marrow to produce multilineage growth factors in vitro. We suggest that these monocyte-derived recruiting activities may play an important role in orchestration of hematopoietic growth factor production by cells of the marrow microenvironment.     

Fibroblast growth factors released by wounded endothelial cells stimulate proliferation of synovial cells.

We demonstrated that vascular endothelial cells mechanically wounded by scraping from the substratum were able to release growth promoting factors for synovial cells as well as for endothelial cells. The wounded endothelial cell conditioned medium stimulated the proliferation of synovial cells derived from different human donors dose dependently and induced transit of growth arrested synovial cells (predominant in the G0 and G1 phases), through the S phase and into the G2 and M phases. When the wounded endothelial cell conditioned medium was applied to heparin-sepharose columns, mitogenic activity was eluted with 2.0 M NaCl. The mitogenic activity in wounded endothelial cell conditioned medium, which was heat sensitive, was inhibited by protamine sulfate and a specific mouse monoclonal antibody against basic fibroblast growth factor (bFGF). Our results are evidence that the wounded endothelial cell conditioned medium contained growth promoting factors for synovial cells, including bFGF. We also showed the presence of bFGF in the endothelial cells of the small blood vessels and stromal synovial fibroblast-like cells in patients with rheumatoid arthritis (RA). Our results suggest that the endothelial cells in the luxuriant capillaries in the synovial tissues from patients with RA may have a critical role in the stimulation of neighboring synovial cell proliferation, resulting in pronounced synovial hyperplasia.     

Ex vivo incubation with growth factors enhances the engraftment of fetal hematopoietic cells transplanted in sheep fetuses.

Hematopoietic stem cells (HSC) transplanted in utero are in competition with endogenous HSC; thus, ultimately the graft constitutes a relatively small fraction of total HSC pool. To enhance the engraftment of donor cells in sheep fetuses, we preincubated these cells, ex vivo, for 16 hours at 37 degrees C with the conditioned medium from phytohemagglutinin-stimulated lymphocytes (PHA-LCM) before in utero transplantation. PHA-LCM is a rich source of hematopoietic growth factors in sheep. Subsequent engraftment was significantly higher in cells preincubated with PHA-LCM compared with fresh cells or those incubated with control medium only. This was reflected in all markers of the donor cells (hemoglobin type, karyotype, and progenitor cell assays). Brief ex vivo incubation with PHA-LCM also increased viability of all marrow cells as well as total numbers of progenitors. Similar enhancement of engraftment was also noted in monkeys after a brief preincubation of donor cells with interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF). We conclude that brief (16 hours) ex vivo incubation of donor cells with a source of such growth factors as IL-3 and GM-CSF enhances the subsequent engraftment of transplanted cells.     

Accelerated cell-cycling of hematopoietic progenitor cells by growth factors

Recent advances in molecular biology have led to the identification of hematopoietic growth factors that support and influence the proliferation of hematopoietic progenitor cells in vitro and in vivo. Although these factors have been extensively studied, little is known of their role in the regulation of cell-cycling of hematopoietic progenitors, especially in the early stage of hematopoiesis. In the present study, we examined the effects of early acting growth factors on proliferative kinetics of hematopoietic progenitors by monitoring the number of cells in individual developing colonies, using an in vitro clonal assay. Interleukin-11 (IL-11) or steel factor (SF), alone or in combination, shortened the time for the size of IL-3-dependent colonies to double. Consecutive replating experiments provided evidence for direct action of growth factors on the growth rate of hematopoietic progenitor cells. Shortening of the time for the total cell number in the colonies to double was due to a reduction in time for each single cell within the respective colonies to become two daughter cells, and there was no alteration in the incidence of cells with a proliferative capacity. Cell-cycle analysis demonstrated that IL-11 has the potential to induce a shortened time for cell-cycle of hematopoietic progenitor cells without affecting distribution of each fraction of the cell- cycle, whereas SF has the potential to reduce cell-cycle time mainly by decreasing the time required for hematopoietic progenitor cells to go through the G1 phase. These results suggest that growth factors may modulate cell-cycling of hematopoietic progenitor cells.     

Cytokines released in vitro by stromal cells from autologous bone marrow transplant patients with lymphoid malignancy

Abstract: Marrow stromal cells of patients treated by autologous bone marrow transplantation (ABMT) for malignancies have been assessed for their ability to secrete granulocyte colony-stimulating factor (G–CSF), granulocyte-macrophage colony-stimulating factor (GM–CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), transforming growth factor β1 (TGFβ1) and macrophage inflammatory protein-1α (MIP-1α). Long-term marrow cultures were established from 10 patients prior to and 3 months after ABMT, from 7 patients 1 yr after ABMT and from 11 controls. Cytokines in culture supernatants of stromal layers (SL) were evaluated by enzyme-linked immunosorbent assay (ELISA). Significant differences between patient groups and controls were apparent in baseline production of GM–CSF, SCF, MlP-1α and TGFβ1. After IL-1β addition in cultures, G–CSF production was reduced in pretransplant and post-transplant patients compared to controls. The production of TGFβ1, LIF, IL-6 and more particularly SCF were reduced in post-transplant patients, while elevated levels of GM–CSF and MIP-1α were observed in these patients only when the values were corrected for the number of cells growing in the SL. These results indicate a prolonged stromal defect in growth factor production following ABMT for the early-stage acting cytokines IL-6, LIF and SCF as well as for G–CSF, but not for GM–CSF, while the production of the 2 inhibitors shows different pathways.     

急性白血病缓解期骨髓造血干/祖细胞体外增殖特征的研究

目的：探讨急性白血病缓解期骨髓造血干／祖细胞增殖特性及对其对不同细胞因子的反应。方法：分离骨髓的单个核细胞（MNC），在含有重组人FLT3配基（rhFL)、干细胞因子（rhSCF)、白细胞介素-3(rhIL-3)、粒－巨噬细胞刺激因子（rhGM-CSF)及促红细胞生成素（EPO）的不同组合下，选用甲基纤维素法半固体培养体系培养7－14d，观察其CFU－GM及BFU－E的集落产率。结果：急性白血病缓期患者骨髓造血干细胞对各种细胞因子增殖反应有所不同（以SCF、FL的作用尤为显著）。与对照组比较，差异有显著性（P＜0．05）。结论：急性白血病缓解期患者的造血干／祖细胞活性比对照组减低。     

Formation of hematopoietic territories and bone by transplanted human bone marrow stromal cells requires a critical cell density

OBJECTIVE: Bone marrow stromal cells (BMSCs) include multipotent cells with the ability to form mature bone organs upon in vivo transplantation. Hematopoiesis in these bone organs has been ascribed to the action of skeletal stem cells, which are capable of differentiating towards bone and hematopoiesis-supporting stroma. Yet, the creation of hematopoietic territories may be in part a natural consequence of the formation of a sufficiently mature and large bone microenvironment. Here, we describe, for the first time, a relationship between BMSC numbers and the extent of bone/hematopoiesis formation in heterotopic transplants. METHODS: Human BMSCs were transplanted along with hydroxyapatite/tricalcium phosphate, utilizing a spectrum of dosages, into immunotolerant mice; the transplants were followed for up to 29 months. RESULTS: The extent of bone and hematopoiesis formation increased with increasing BMSC numbers; however, the relationship was sigmoid in character, and a threshold number of BMSCs was necessary for extensive bone formation or any hematopoiesis. Hematopoiesis only occurred in conjunction with extensive bone formation, and no hematopoiesis occurred where bone formation was poor. Consistent with our earlier studies of long-term BMSC transplantation, the transplants underwent a change in bone morphology but not bone content after 8 weeks. CONCLUSION: Our results have provided evidence that the formation of both hematopoiesis and a mature bone organ is as much a consequence of a sufficiently high local density of bone marrow stromal cells as it is the product of skeletal stem cell action.     

Comparative proteomic characterization of articular cartilage tissue from normal donors and patients with osteoarthritis

OBJECTIVE: To identify potential molecular mediators and biomarkers for osteoarthritis (OA), through comparative proteomic analysis of articular cartilage tissue obtained from normal donors without OA (n = 7) and patients with OA (n = 7). METHODS: The proteomic analyses comprised extraction of soluble proteins from cartilage, separation of the protein mixtures by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by in-gel digestion, and subsequent nano-liquid chromatography-tandem mass spectrometry analysis in conjunction with a database search for protein identification and semiquantitation. RESULTS: A total of 814 distinct proteins were identified with high confidence from 14 samples; 420 of these proteins were detected with > or = 3 unique peptides in at least 4 samples from the same group. Using stringent criteria, 59 proteins were found to be differentially expressed in OA cartilage. Gene Ontology and Ingenuity pathway analysis tools were used to characterize these proteins into functional categories. One of the up-regulated proteins, HtrA1, a serine protease, was detected at high levels in cartilage. CONCLUSION: Altered protein expression in the disease state is associated with many aspects of the pathogenesis of OA, such as increased proteolysis, lipid metabolism, immune response, and decreased signal transduction. To our knowledge, this is the first time that a large portion of these proteins and their expression patterns were identified in cartilage, thus providing new insights for finding novel pathologic mediators and biomarkers of OA.     

Adverse effects of nutritional deprivation on transplanted hematopoietic cells.

We studied the effect of food deprivation on hematopoietic reconstitution of B6D2F1 mice given 900 rad total body irradiation followed by 2 x 10(5) syngeneic bone marrow cells. Animals deprived of food from the day of cell transfer to the day of sacrifice were compared to control animals allowed ad libitum laboratory chow. The body weight of food deprived mice decreased by 36% on day 7 as compared to a 9% decrease in fed controls. The mean number of nucleated cells/femur on day 7 was only 22% of that found in fed controls. The spleen weight in the experimental animals was only 48% of that in the controls. Food deprived animals showed complete suppression of macroscopic hematopoietic spleen colony formation. Both marrow and spleen from the primary recipients, when studied for content of CFU-s in secondary ad libitum fed recipients, showed that food deprived animals had less than 25% of the number seen in controls. A third group of animals receiving vitamin supplements and small amounts of dextrose, but no protein, showed hematopoietic suppression similar to that seen in the totally food deprived mice.     

Trends in transplantation of hematopoietic stem cells from unrelated donors

Transplants of hematopoietic stem cells from unrelated donors have become feasible for patients with a growing variety of hematologic disorders. The probability of finding a suitable donor has increased because of the expansion of the network of registries containing more than seven million HLA-typed donors worldwide. The selection of compatible donors has become more effective, thanks to the discovery of new HLA alleles and the development of precise and efficient HLA typing methods using DNA technology. Improved methods for transplantation may provide the opportunity to further decrease treatment-related toxicity and improve survival.     

Crowding-dependent production of colony-stimulating factors by cultured syngeneic or allogeneic hematopoietic cells

Mitogenic stimulation in vitro of mouse T lymphocytes induces the production of the hematopoietic cytokines granulocyte-macrophage colony-stimulating factor and IL-3. The present experiments showed that simple crowding of murine spleen or lymph node cells was a sufficient inducing stimulus. Crowding did not have this consequence for thymus or marrow cells or spleen cells from nu/nu or Rag-1-/- mice lacking T lymphocytes. Crowding for as short a period as 24 h was sufficient to allow subsequent cytokine production in sparse cultures. Purified T lymphocytes also exhibited low levels of crowding induction of cytokine production and cytokine production was enhanced by IL-2 and IFN-gamma. However, IFN-gamma-/- spleen cells exhibited similar crowding-induced colony-stimulating factor production to that of control spleen cells. Excess cell crowding inhibited cytokine production. This inhibition was not caused by receptor internalization of cytokines but may contribute to the failure to observe IL-3 production in lymphoid organs in vivo. Coculture of allogeneic spleen or peritoneal cells was a strong inducing signal for colony-stimulating factor production but this parameter was unable to detect autoreactivity of T lymphocytes in mice that lack suppressor of cytokine signaling 1 and exhibit T lymphocyte activation.     

Consequences of extremely high doses of irradiation on bone marrow stromal cells and the release of hematopoietic growth factors

Although hematopoietic growth factors have previously been difficult to demonstrate in long-term murine bone marrow cultures, it is possible to demonstrate release of growth factors from adherent cells of these long-term cultures following modest doses of irradiation. The present studies were undertaken to determine the maximally tolerated dose of irradiation for growth factor-producing stromal cells and to characterize the growth factor activities. It was discovered that stromal cells could survive extremely high doses of irradiation (500 Gy) and continue to elaborate hematopoietic growth factors. Using escalating doses of irradiation, a dose-dependent increment in detectable hematopoietic growth factors was detectable in unconcentrated conditioned medium. Conditioned medium from long-term cultures exposed to 500 Gy stimulated both fresh murine bone marrow cells (15 +/- 2 to 81 +/- 5 CFU-C/5 X 10(4) target cells) and the interleukin-3/GM-CSF-responsive cell line FDC-P1. In the CFU-C assay, this activity appeared to be predominantly monocyte/macrophage differentiating activity (M-CSF), based upon colony morphology. However, following stimulation of these irradiated stromal cells with endotoxin, there was a significant increase in FDC-P1 growth-promoting activity, and in the CFU-C assay there was increased production of granulocyte, megakaryocyte, and blast-cell type colonies indicating the increased release of a multilineage growth factor. The stromal cells surviving these extremely high doses of irradiation represent a heterogeneous population as demonstrated by morphologic, histochemical, and functional characterization. The two predominant cell populations included a macrophage-like cell and a large flat cell previously referred to as a "blanket" cell.     

Collection of peripheral blood stem cells from normal donors 60 years of age or older

We report 14 normal peripheral blood stem cell (PBSC) donors ≥ 60 years of age who had cytokine mobilization followed by PBSC apheresis for allogeneic transplantation. Mobilization was achieved with filgrastim (6 μg/kg twice daily). Their median age was 63.5 years (range 60–77), and 43% had a positive medical history, mainly hypertension and/or cardiac problems. Their median pre-apheresis leucocyte count (×10⁹/l) was 38.6 (range 29.6–63.4). The median apheresis yield (×10⁶ CD34⁺ cells/litre blood processed, first apheresis) was 27.9 (range 1.6–54.8). The target cell dose (≥4& times; 10⁶ CD34⁺ cells/kg recipient) was reached with one procedure in eight (57%) donors. Filgrastim-related adverse events were acceptable and apheresis was well tolerated. When compared to younger donors (<60 years of age), a trend to a lower CD34⁺ apheresis yield and a requirement for more than one apheresis to achieve the collection target (≥4 ×10⁶ CD34⁺ cells/kg) was evident. Although older (≥60 years) donors seem to mobilize less effectively, these data suggest that PBSC collection from them is feasible and has an acceptable short-term safety profile.     

Expansion of haematopoietic stem cells from normal donors and bone marrow failure patients by recombinant hoxb4

In this study six versions of recombinant human hoxb4 proteins were produced and their effectiveness evaluated in expanding human haematopoietic stem and progenitor cells in vitro and in vivo . An N-terminal-tat and C-terminal histidine-tagged version of hoxb4 (T-hoxb4-H) showed the highest activity in expanding colony forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) when used at 50 nmol/l concentration in cell culture. Human cord blood CD34⁺ cells cultured with 50 nmol/l T-hoxb4-H showed a significant increase in severe-combined immunodeficient mouse-repopulating cells (SRCs). In a mouse model of immune-mediated bone marrow (BM) failure, T-hoxb4-H showed an additive effect with cyclosporine in alleviating pancytopenia. In addition, T-hoxb4-H expanded CFC and LTC-IC on BM samples from patients with refractory severe aplastic anaemia and myelodysplastic syndromes: after culturing with 50 nmol/l T-hoxb4-H for 4 d, BM cells from 10 of the 11 patients showed increases in CFC and LTC-IC, and the increase in LTC-IC was statistically significant in samples from four patients. Recombinant human hoxb4 could be a promising therapeutic agent for BM failure.