Characterization of bone marrow mesenchymal stromal cells in aplastic anaemia

In aplastic anaemia (AA), haemopoietic activity is significantly reduced and generally attributed to failure of haemopoietic stem cells (HSC) within the bone marrow (BM). The regulation of haemopoiesis depends on the interaction between HSC and various cells of the BM microenvironment, including mesenchymal stromal cells (MSC). MSC involvement in the functional restriction of HSC in AA is largely unknown and therefore, the physical and functional properties of AA MSC were studied in vitro. MSC were characterized by their phenotype and ability to form adherent stromal layers. The functional properties of AA MSC were assessed through proliferative, clonogenic and cross‐over culture assays. Results indicate that although AA MSC presented typical morphology and distinctive mesenchymal markers, stromal formation was reduced, with 50% of BM samples failing to produce adherent layers. Furthermore, their proliferative and clonogenic capacity was markedly decreased (P = 0·03 and P = 0·04 respectively) and the ability to sustain haemopoiesis was significantly reduced, as assessed by total cell proliferation (P = 0·032 and P = 0·019 at Week 5 and 6, respectively) and clonogenic potential of HSC (P = 0·02 at Week 6). It was concluded that the biological characteristics of AA MSC are different from those of control MSC and their in vitro haemopoiesis‐supporting ability is significantly reduced.     

Human marrow stromal cells in short-term semi-solid bone marrow culture in aplastic anaemia

A short-term methylcellulose technique was used to study the proliferation of marrow-derived stromal, erythroid and myeloid colonies from normal controls and from patients with aplastic anaemia. There was a significant reduction in all colony types in patients with aplastic anaemia when compared with normal controls. In 4 patients who achieved remission following treatment with ATG + oxymetholone or with oxymetholone alone there was a return to normal range of stromal colonies and also of CFU-E. There was no change in the numbers of BFU-E's and only a slight increase in the numbers of CFU-C's.     

Correction of stromal cell defect after bone marrow transplantation in aplastic anaemia

Defects in stromal cell function have been demonstrated in a number of aplastic anaemia (AA) patients. Here we have studied a patient with severe AA and abnormal stromal cell function who underwent bone marrow transplantation (BMT). The objective of this study was to investigate the timing and the mechanism of correction of the stromal defect after transplantation. The patient, a 25-year-old woman with severe AA, underwent BMT from her brother. BM was obtained from the patient on five occasions: 2 weeks pre BMT, and 3, 8, 16 and 21 months post BMT. Stromal cells were grown to confluence and recharged with purified CD34+ cells from normal donors. The support of such cells, as assessed by weekly colony-forming assay (CFU) of non-adherent cells, was compared with that of stromal layers grown from normal BM. A novel technique of combined fluorescence in situ hybridization (FISH) and immunocytochemistry was used to determine the origin of specific stromal cell types on cytospins of stroma post BMT. Stromal function was defective at 2 weeks pre BMT and at 3 months post BMT, but returned to normal at 8 and 16 months post BMT. At 21 months post BMT, stromal fibroblasts and endothelial cells were shown to be of recipient origin, and macrophages and T cells were of donor origin. We present here evidence in a case of severe AA for defective stromal function before BMT and delayed normalization of function after BMT. This correlated with engraftment of donor macrophages and T cells, but not fibroblasts and endothelial cells.     

Functionally abnormal marrow stromal cells in aplastic anemia

We studied the myeloid colony-stimulating activity (CSA) of marrow stromal cells (MSC) derived from normal subjects and patients with aplastic anemia, acute leukemia, and other myeloproliferative disorders. CSA of the MSC was determined in a bilayer system. Feeder layers with varying numbers of MSC (10(4) to 2 X 10(5)) were used. Of 40 MSC tested, 39 stimulated myeloid colony formation by the normal target marrow mononuclear cells. The optimal concentration of MSC exhibiting the maximal stimulation of myeloid progenitors (CFU-GM) varied with different MSC. MSC from normal subjects and from patients with acute leukemia and myeloproliferative disorders were potent stimulators of CFU-GM differentiation. In contrast, MSC from patients with aplastic anemia had poor CSA, suggesting that the marrow microenvironment is functionally abnormal in aplastic anemia.     

In vitro assessment of marrow ''stem cell''and stromal cell function in aplastic anaemia

An in vitro model system is described that allows separate assessment of 'stem cell' and stromal cell function in aplastic anaemia (AA). Seven patients with non-severe AA, who had responded to immunosuppressive therapy and had haematological evidence of residual marrow function, were studied. Of these, three with otherwise typical AA had an acquired clonal cytogenetic marker. Purified bone marrow haemopoietic progenitors labelled with CD34 monoclonal antibody were positively selected using the fluorescence activated cell sorter (FACS) from both normal subjects and from patients with AA. The generative capacity of the CD34 positive cells was assessed by monitoring the output of granulocyte/macrophage colony forming cells (CFU-GM) in the non-adherent layer after inoculation onto irradiated performed long-term marrow culture (LTBMC) stromas. Stromal function in AA was assessed by inoculating CD34 positive cells from normal bone marrow onto performed irradiated stromas from patients with AA. Haemopoietic cell ('stem cell') function in AA was assessed by inoculating CD34 positive cells from AA patients onto confluent irradiated normal marrow stromas. Using these crossover/LTBMC experiments, all patients exhibited severe defects in haemopoietic cell function with normal functioning stroma. The proportion of CD34 positive cells present in bone marrow from these patients was reduced compared with controls, they comprised fewer small primitive 'blast-like' cells which in normal bone marrow are known to possess marrow repopulating ability, and demonstrated reduced clonogenic potential in short-term colony assays.     

Plasma levels and production of soluble stem cell factor by marrow stromal cells in patients with aplastic anaemia

Defective marrow stroma or microenvironment have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). Stem cell factor (SCF), the ligand for the c-kit receptor, is produced mainly by marrow stromal cells and seems to reflect the haemopoietic function of bone marrow stroma. We measured the plasma levels of soluble SCF in 87 patients with AA and investigated the production of soluble SCF by the marrow stromal cells of 46 patients with acquired AA. The mean plasma SCF concentrations in the AA patients and normal controls were not significantly different (1098 ± 398 pg/ml versus 1160 ± 316 pg/ml, respectively), and there was no significant correlation between the peripheral blood counts and the SCF concentrations. However, the mean SCF concentration in patients who received prednisolone ± anabolic steroids at the time of sampling was significantly lower than that in the patients who did not receive both agents. We did not find any correlation between the changes in SCF concentrations and the response to immunosuppressive therapy, although it did increase significantly after bone marrow transplantation. The ability of marrow stromal cells to release soluble SCF did not differ significantly between the patients with AA and normal controls. We conclude that soluble SCF production does not appear to be altered in patients with AA and that defective production of soluble SCF is unlikely to be the cause of AA in most patients.     

Increased apoptotic cells in bone marrow biopsies from patients with aplastic anaemia

In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anaemia (AA) we determined the proportion of apoptotic cells in paraffin-embedded bone marrow biopsies from patients with aplastic anaemia using an in situ TdT-catalysed DNA nick end labelling (TUNEL) staining method. A significant increase in the proportion of mononuclear apoptotic cells was demonstrated in biopsies from patients with aplastic anaemia (8.19 ± 1.45%) when compared with controls (2.07 ± 0.86%). These data support the view that apoptosis may play a role in the pathophysiology of bone marrow failure.     

Long-term culture of aplastic anaemia bone marrow

Long-term bone marrow cultures (LTBMC) were established with marrow from 11 patients with aplastic anaemia (AA). Bone marrow from five patients, with low numbers of committed progenitor cells, exhibited an increase in committed progenitor cell production to normal levels in the first week of LTBMC. None of 44 haematologically normal marrow cultures showed this increase. Mature and committed progenitor cell production in all cultures from aplastic anaemia bone marrow, declined faster than in normal cultures. This study indicates that short-term culture for committed progenitor cells is an underestimate of the proliferative capacity of bone marrow from some patients with AA. LTBMC may provide a useful system for further studies into the mechanisms responsible for this increased growth in some patients with AA.     

A proliferative defect of marrow cells in experimental chronic hypoplastic marrow failure (aplastic anaemia)

Marrow cells from control mice and mice with chronic hypoplastic marrow failure (CHMF, aplastic anaemia) were grown in tissue culture and growth was assessed by measuring the number of cells/colony and uptake of 3H-thymidine/colony. Cells from mice with CHMF showed impaired proliferation in response to colony stimulating factor. Mixing experiments suggested that the impairment of proliferation was not dut to alteration in suppressor or helper cells but to an intrinsic lesion of the marrow cells themselves.     

Telomere length changes in patients with aplastic anaemia

To investigate telomere changes in patients with aplastic anaemia (AA) and clinical factors influencing the telomere dynamics, telomere length (TL) was measured in peripheral blood mononuclear cells using Southern blot analysis of 42 patients with AA and 39 healthy normal controls. Nineteen patients received supportive treatment only, while the remaining 23 patients received immunosuppressive therapy with anti-thymocyte globulin or anti-lymphocyte globulin +/- cyclosporin A. In AA patients, TL was on average 1.41 kb shorter than that of age-matched normal controls (P < 0.001). In patients treated with immunosuppression, the mean TL of non-responders was significantly shorter than that of age-matched normal controls (P < 0.001), while no difference in TL was detected in responders compared with controls. Positive correlation was observed between the extent of telomere shortening, the severity of neutropenia (P = 0.05) and the degree of mean corpuscular volume elevation (P = 0.005) at the time of the study. However, there was no correlation with time elapsed since diagnosis (P = 0.214). These findings suggest that haematopoietic stem cells in patients with AA rapidly lose TL at the onset of the disease. The TL shortening may reflect the severity of impairment of haematopoiesis.     

Selective reduction of natural killer T cells in the bone marrow of aplastic anaemia

T cell-mediated suppression of haematopoiesis is believed to play an important role in the pathophysiology of aplastic anaemia (AA) and in the pancytopenia of some myelodysplastic syndromes (MDS). Natural-killer T (NKT) cells belong to a unique lymphocyte subset that expresses an invariant T-cell receptor (TCR), consisting of Valpha24JalphaQ, and common NK cell surface markers. NKT cells have been hypothesized to play a role in immune regulation, and many human autoimmune conditions are associated with NKT cell deficiency. Here we investigate the role of NKT cells in AA and MDS patients. Flow cytometry demonstrated that NKT cells, unlike other T-lymphocyte subpopulations, were disproportionally decreased in AA and MDS marrow. When we compared variability within the CDR3 region of Valpha24 in CD4-CD8- T cells derived from AA and healthy individuals, the CDR3 size of Valpha24 cells showed a polyclonal distribution in AA patients, while in control subjects a typical oligoclonal or monoclonal pattern was found. Southern blot and sequence analysis of Valpha24 polymerase chain reaction products revealed that the NKT cell-specific JalphaQ region was predominant in control subjects, whereas it was not, or only very weakly, detected in AA and MDS patients. These results show that NKT cells are profoundly decreased in AA and MDS, and their deficiency may, as in other human autoimmune diseases, play a role in the local immune dysregulation in AA and MDS.     

Improved survival following bone marrow transplantation for aplastic anaemia

We transplanted 46 patients with severe aplastic anaemia with a new pretransplant immunosuppressive regimen consisting of cyclophosphamide (200 mg/kg) and low-dose total body irradiation (3 Gy). This regimen (CY-TBI-2) was designed to decrease the high risk of graft rejection associated with the use of cyclophosphamide alone, without increasing the incidence of graft-versus-host disease (GHVD) or interstitial pneumonia (IPn). Two-year actuarial disease-free survival of patients conditioned with CY-TBI-2 was 62% (95% CI: 47-77%). Only one patient rejected her graft and the incidence and severity of GVHD and IPn were not increased compared to previous studies. Patients less than 25 years of age had excellent 2-year survival of 82% (95% CI: 69-95%). These data indicate that CY-TBI-2 is an effective means of preventing graft-rejection and achieving long-term disease-free survival in multiply transfused patients with severe aplastic anaemia.     

Decreased frequency of bone marrow NK progenitors in aplastic anaemia

Twelve patients with aplastic anaemia were studied with regard to the frequency of NK progenitors in the bone marrow (BM) to investigate the mechanism of depressed NK activity and low NK cell count in the peripheral blood. NK cell (CD16+ cell) count and NK (K562) activity were significantly decreased (P less than 0.02 and P less than 0.001, respectively) in the patients as compared to eight healthy control subjects. Nylon wool non-adherent (NW-NA) BM mononuclear cells (MNC) of each patient and control were prepared. Mature T and NK cells were extensively depleted by sheep red cell rosette formation followed by a centrifugation on Ficoll-Hypaque and monoclonal antibody mediated complement dependent cytolysis. Those selected BM cells were cultured in the presence of recombinant interleukin 2 (rIL2). Generation of NK activity was significantly decreased (P less than 0.01) in the patients with aplastic anaemia. Frequency of NK progenitors in the selected BM cells assayed by the limiting dilution method was significantly decreased in those patients (P less than 0.05). The frequency of BM NK progenitors relative to NW-NA BM cells were related to NK cell count (P less than 0.01). Those results indicate that depressed NK activity in aplastic anaemia is closely related to decreased NK cell count which is probably due to decreased production of NK cells in the BM.     

Late outcomes after bone marrow transplant for aplastic anaemia

Allogeneic transplantation is effective in reconstituting haemopoiesis in severe aplastic anaemia (SAA). We report long-term health-related outcomes in 37 children and young adults with SAA transplanted between 1975 and 1996. The median length of follow-up was 17 years (range, 4-25 years). Using a case-control design, late social and medical outcomes in transplant recipients were compared with 146 control subjects matched for gender and age. The majority of patients received an irradiation-containing preparative regimen. There were no significant differences in the self-rating of health status between transplant recipients and controls (P = 0.8), with 71% reporting their health status as excellent and 29% as good compared with 74% and 26% of controls. They demonstrate the same normal psychosexual function as their peers and have similar educational achievements and employment history. Transplant recipients and controls are equally likely to have held a job or be currently employed and there are no significant differences in their personal income (OR = 0.60, 95% CI = 0.11-3.37). Although transplant recipients have had problems related to health insurance policies, the majority have adequate health insurance coverage. There were no differences in chronic health problems between transplant recipients and control subjects, except for expected increases in cataracts, short stature in men, hypothyroidism and gonadal dysfunction. Using self-assessment, these transplant recipients indicated an excellent level of satisfaction and social integration, showing transplantation to be an effective long-term therapy for SAA.     

Regeneration of peripheral blood cells following allogeneic bone marrow transplantation for severe aplastic anaemia

S ummary . We have studied the pattern of regeneration of peripheral blood cells following ABO compatible bone marrow transplantation for severe aplastic anaemia. 18 patients were treated with cyclosporin A and six with methotrexate for post graft immunosuppression. The number of days taken for the neutrophil count to reach 0.5 × 10⁹/l, the lymphocyte count to reach 1.0 x 10⁹/l, the platelet count to reach 100 × 10⁹/l and the reticulocytes to reach 1% was shorter in the CyA treated patients. This finding reached statistical significance for all types of cells except the platelets (neutrophils, P < 0.001; lymphocytes, P < 0.02; platelets, P > 0.05; reticulocytes, P < 0.001).     

Bone marrow fibroblast function in relation to granulopoiesis in aplastic anaemia

Fibroblasts grown from the bone marrow of normal individuals and incubated with colony-stimulating factor (CSF) enhance the stimulating activity of this CSF on granulocyte-macrophage colony-forming cell (GM-CFC) proliferation. For this study, the ability of fibroblast monolayers grown from the marrows of six patients with severe aplastic anaemia to increase the activity of CSF has been compared with the activity of normal fibroblasts. Tests of this function showed subnormal CSF-enhancing activity by fibroblasts grown from the marrows of three of the six aplastic patients. Since cultured bone marrow fibroblasts are thought to represent an important component of the haemopoietic microenvironment, the results suggest that some of the patients had a microenvironmental abnormality at the time of study. One of the patients whose fibroblasts were abnormal was reinvestigated after he had been given a graft of allogeneic bone marrow cells. His post-graft fibroblast function was normal, showing that the abnormality was reversible.     

Stem cells and the microenvironment in aplastic anaemia

Normal blast colony-forming cells (BI-CFC) bind to stroma cultured in the presence of methylprednisolone (MP⁺) but not to MP^- stroma. In aplastic marrow, the incidence of BI-CFC is variable (0–4 x normal values) and there is no consistent relationship with the CFU-GM (granulocyte-macrophage colony-forming cell) content. Normal stroma require MP to induce BI-CFC binding function and form fat cells whereas MP^- stroma grown from 4/9 aplastic patients formed fat cells and bound BI-CFC. The 5/9 aplastic cases that did not form fat cells spontaneously also bound BI-CFC moderately better than normal stroma. This suggests that the haemopoietic microenvironment in aplastic anaemia responds physiologically to bone marrow failure by increasing its haemopoietic support capacity.     

Myelopoiesis and erythropoiesis of bone marrow cells cultured in vitro in patients recovered from aplastic anaemia

Methylcellulose culture assay was used to detect committed haemopoietic stem cells, CFU-C and CFU-E, in aplastic anaemia patients with autologous haemopoietic reconstitution. Severe diminution of CFU-C was found in all the patients studied and the absence of a dose-response to colony stimulating factor (CSF) was demonstrated. A reduced number of CFU-E and lower erythropoietin (Ep) sensitivity of those progenitors was detected as well. Autologous serum added to the bone marrow cultures of these patients enhanced the growth of CFU-C but inhibited CFU-E growth. According to the results presented, some residual damage at the stem cell level is suggested.