The tale of early hematopoietic cell seeding in the bone marrow niche

Since introduction of the notion of a "niche" that hosts engraftment and activity of hematopoietic cells, there is a massive effort to discover its structure and decipher its function. Our understanding of the niche is continuously changing with reinterpretation of traditional concepts and apprehension of new insights into the biology of hematopoietic cell homing, seeding, and engraftment. Here we discuss some of the early events in hematopoietic stem cell seeding and engraftment and propose a perspective based on visualization of labeled bone marrow cells in real time in vivo. Primary seeding of hematopoietic cells in the bone marrow niches evolves as a complex and dynamic process; however, it follows discrete topological and chronological patterns. Initial seeding occurs on the endosteal surface of the marrow, which includes heterogeneous niches for primary seeding. Several days after transplantation the endosteal niches become more restrictive, hosting primarily mitotically quiescent cells, and gradual centripetal migration is accompanied by engagement in proliferation and differentiation. The hematopoietic niches evolve as heterogeneous three-dimensional microenvironments that are continuously changing over time.     

Stable mixed hematopoietic chimerism after bone marrow transplantation for sickle cell anemia.

A multicenter investigation of allogeneic bone marrow transplantation for children with sickle cell disease was conducted that included 27 European and North American transplant centers. Fifty-nine patients who ranged in age from 3.3 to 15.9 years (median, 10.1 years) received HLA-identical sibling marrow allografts between September 1991 and April 2000. Fifty-five patients survive, and 50 survive free from sickle cell disease, with a median follow-up of 42.2 months (range, 11.8 to 115 months) after transplantation. Of the 50 patients with successful allografts, 13 developed stable mixed donor-host hematopoietic chimerism. The level of donor chimerism, measured > or =6 months after transplantation in peripheral blood, varied between 90% and 99% in 8 patients. Five additional patients had a lower proportion of donor cells (range, 11% to 74%). Among these 5 patients, hemoglobin levels varied between 11.2 and 14.2 g/dL (median, 11.3 g/dL; mean, 12.0 g/dL). In patients who had donors with a normal hemoglobin genotype (Hb), the Hb S fractions were 0%, 0%, and 7%, corresponding to donor chimerism levels of 67%, 74%, and 11%, respectively. Among patients who had donors with sickle trait, the Hb S fractions were 36% and 37%, corresponding to donor chimerism levels of 25% and 60%, respectively. Thus, allograft recipients with stable mixed chimerism had Rb S levels similar to donor levels, and only 1 patient required a red blood cell transfusion beyond 90 days posttransplantation. None of the patients have experienced painful events or other clinical complications related to sickle cell disease after transplantation. These observations strongly suggest that patients with sickle cell disease who develop persistent mixed hematopoietic chimerism after transplantation experience a significant ameliorative effect.     

Histopathological bone marrow changes after reduced-intensity hematopoietic stem cell transplantation for follicular lymphoma involving bone marrow

Allogeneic stem cell transplantation (allo-SCT) is used as curative therapy for malignant lymphoma, and reduced-intensity hematopoietic stem cell transplantation (RIST) is sometimes performed to avoid the toxicity and mortality associated with myeloablative allo-SCT. RIST is generally preferred for elderly patients with malignant lymphoma. A 62-year-old woman with follicular lymphoma (FL) involving bone marrow (BM) suffered relapse after autologous SCT. RIST was performed; cells were from an unrelated, fully human leukocyte antigen-matched donor. To study the hematopoietic reconstitution, BM biopsy specimens that were obtained at different times after RIST, were evaluated. Engraftment of donor cells was observed on days 19 and 48 after RIST, and residual FL in BM had completely disappeared by day 73 after RIST. This is the first report to document histological BM regeneration after RIST and disappearance of FL involving the BM.     

T cell regeneration after allogenic bone marrow transplantation

Various T cell subsets were characterized by double immunofluorescent staining using monoclonal antibodies (MoAb) in blood, bone marrow (BM) and tissues of 29 patients after allogeneic BM transplantation (BMT). In an attempt to prevent graft versus host disease (GvHD), 15 patients received cyclosporin A (Cy A). In the remaining 14 patients the BM was pre-incubated with a MoAb, OKT3. The regeneration of T4⁺ subset was delayed and the level of T8⁺ cells was abnormally high even 1 year after engraftment. This did not have any predictive value for the appearance of complications such as GvHD or severe viral infections. The number of T8⁺ cells was lower in the group of patients who received Cy A than in the OKT3 group (0·7±0·2 vs 1·5±0·3×10⁹/1 at day 90). In contrast to normal individuals, the T4/T8 ratio in both blood and regenerating BM of BMT patients was <1. A sizeable subset of circulating T cells expressed the phenotype T8⁺,T10⁺,HNK-1⁺,DR⁺. Circulating cells of this phenotype were transiently very high (up to 50%) in patients with active GvHD or suffering from severe viral infection. This subpopulation of lymphocytes was not found in the epidermal infiltrate that accompanied GvHD where the predominant phenotype was T8⁺,T1^-,T10^-,HNK-1^-,DR^-. We conclude therefore that after BMT the number and phenotype of circulating T cells reflects the T cell distribution seen in the regenerating BM.     

Immunologic and hematopoietic effects of recombinant human prolactin after syngeneic bone marrow transplantation in mice.

The period of immune deficiency following bone marrow transplantation (BMT) results in a susceptibility to opportunistic infections and remains a growing obstacle in improving the efficacy of BMT. Neuroendocrine hormones have been shown to affect numerous immunologic and hematologic responses after in vivo administration. We investigated whether neuroendocrine hormones, notably prolactin (PRL), could be administered after BMT and result in improved immunologic recovery. Mice were given lethal total body irradiation followed with a congeneic or a syngeneic BMT. Some groups then received recombinant human PRL (rhPRL) daily for 3 weeks. Effects on immune reconstitution and function were then monitored. The results show that PRL could increase thymic cellularity and donor T-cell reconstitution after congeneic BMT. Increases in B cells and myeloid progenitors were also observed. Mitogenic responses by both T and B cells were observed after PRL treatment. These results suggest that PRL may be of use to promote immune and myeloid reconstitution after BMT.     

Morphology of the bone marrow after stem cell transplantation

In many haematological conditions the only curative option is stem cell (SCT) or bone marrow (BM) transplantation. Little information exists about BM morphology following non-ablative engraftment. During the pretransplantation period and depending on the kind of pretreatment, there may be hypoplasia, residual disease and varying degrees of fibrosis. In the post-transplantation period, after 1-3 weeks of transfusion-dependent pancytopenia, the first signs of successful engraftment are indicated by the recurrence of neutrophils, monocytes and erythrocytes in the peripheral blood. In the BM there is slow regeneration of erythropoiesis, followed by the other lineages of haematopoiesis and increase in reticulin fibres or even a resolution of fibrosis. Diagnostic problems arise when neoplastic lympho- or haematopoiesis are maintained following transplantation. Moreover, there may be a significant graft versus tumour response reaction or an already relapsing disease needing aggressive treatment. On the other hand, a conspicuous dyshaematopoiesis should not be mistaken as representing a myelodysplastic syndrome. The presence of granulomas being treatment-related or a manifestation of intercurrent granulomatous disease has to be considered. More advanced knowledge of the histological features of regenerating BM will certainly aid the recognition of relapsing disease and is needed for the adequate reporting of post-transplant alterations associated with a successful or failing engraftment.     

Graft versus host disease following allogeneic bone marrow transplantation.

Graft versus host disease is a major barrier in allogeneic bone marrow transplantation. The associated morbidity and mortality need to be understood and prevented if possible, as the potential indications for bone marrow transplantation continue to broaden. Areas of investigation include the cellular effector arm as well as the cytokines associated with the expression of the disease.     

Kinetics of erythrogenesis after bone marrow transplantation.

To determine the kinetics of bone marrow erythrogenesis after bone marrow transplantation, the authors counted reticulocytes (by blood smear and flow cytometry) and compared those data with neutrophil and platelet recovery in 23 consecutive bone marrow transplant patients. The earliest indication of marrow recovery after allogeneic and autologous bone marrow transplantation was defined as the second increasing cell count after the lowest recorded count, provided that the trend continued upward. Recovery of marrow function was detected earlier in 10 of 23 patients using reticulocyte counts than by either neutrophil or platelet count alone. Specifically, in 8 of these 10 patients, recovery of erythropoiesis was determined earlier by flow cytometric examination than by the blood smear method. On the other hand, combining the data using the earliest value of platelet, neutrophil, and reticulocyte counts indicated that the mean day of recovery in our patient population was determined to be 12.1 +/- 4 days after marrow infusion. In patients undergoing autologous and allogeneic bone marrow transplantation, serial neutrophil and reticulocyte count determinations are complementary in early clinical detection of successful engraftment.     

Problems of infection after bone marrow transplantation.

Representatives of 17 bone marrow transplant (BMT) teams met to discuss the problems of infection after BMT. With experience of more than 2200 transplanted patients over the past 10 yr, the changes in the patterns of infection were surprisingly similar. Deaths due purely to bacterial infection have greatly diminished and the major early problems today result from fungi and cytomegalovirus. The role of non-herpes group viruses has only recently received attention. With increasing numbers of survivors, late bacterial infections are assuming importance. The meeting also provided an opportunity to document the measures adopted to prevent infection during BMT and following discharge.     

Increased serum IgE concentrations during infection and graft versus host disease after bone marrow transplantation.

Serum IgE concentrations estimated in 25 bone marrow transplant recipients during episodes of infection or graft versus host disease, or both, were raised not only in some patients with acute graft versus host disease but also in many patients with infection. Raised values were not seen in chronic graft versus host disease. The routine estimation of serum IgE in bone marrow transplant recipients had minimal value because of the lack of specificity of the IgE response.     

Remodeling of the thoracic aorta after bone marrow cell transplantation

Stem cells are characterized by their ability to differentiate into multiple cell lineages and display the paracrine effect. The aim of this work was to evaluate the effect of therapy with bone marrow cells (BMCs) on blood glucose, lipid metabolism and aortic wall remodeling in mice through the administration of a high fat diet and subsequent BMCs transplantation. C57BL/6 mice were fed a control diet (CO group) or an atherogenic diet (AT group). After 16 weeks, the AT group was divided into four groups: an AT 14 days group and AT 21 days group, that were given an injection of vehicle and sacrificed at 14 and 21 days after, respectively; AT-BMC 14 days group and AT-BMC 21 days group that was given an injection of BMCs and sacrificed at 14 and 21 days after. The CO group was sacrificed along with other groups. The BMCs transplant had reduced blood glucose, triglycerides and total cholesterol. The Qa (1/mm²) was quantitatively reduced in AT 14 days group, AT 21 days group and was high in AT-BMC 21 days group. The AT 21 days group exhibited increased tunica media and elastic system fibers. The immunolabeling for α-SMA and VEGF showed less immunolabeling in transplanted groups with BMCs. The immunostaining for PCNA seems to be more expressive in the group AT-BMC 21 days group. To conclude, our results support the concept that in mice, the injection of BMCs improve glucose levels, lipid metabolism and remodeling of the aortic wall in animals using atherogenic diet.     

Autologous bone marrow transplantation after histologic transformation of indolent B cell malignancies.

The role of high-dose therapy and autologous stem cell transplantation in diffuse large B cell lymphoma (DLBCL) after transformation is controversial. We have retrospectively analyzed patients with chemosensitive disease and a history of follicular lymphoma or chronic lymphocytic leukemia/small lymphocytic lymphoma who underwent high-dose chemoradiotherapy and bone marrow transplantation (BMT) with anti-B cell monoclonal antibody-purged autologous marrow for DLBCL. Between December 1982 and August 1997, 27 patients underwent autologous BMT using a uniform ablative regimen with cyclophosphamide, total-body irradiation, and bone marrow purging. All patients received multiple chemotherapy regimens before autologous BMT. At bone marrow (BM) harvest, only 44% of patients were in complete remission, and overt BM infiltration was present in 37%. After cyclophosphamide and total-body irradiation, no treatment-related deaths were seen. Eleven of the 27 patients relapsed, and four patients developed myelodysplasia/acute myelogenous leukemia. In seven patients in whom pathologic studies were available after relapse, the histology remained DLBCL. Twelve patients remained alive and in complete remission with a median follow-up of 36 months (range 10-132). The disease-free survival and overall survival are estimated to be 46% (90% confidence interval 28-64) and 58% (40-76) at 5 years, respectively. Patients whose disease underwent histologic transformation within 18 months of their initial diagnosis of indolent lymphoma had significantly better overall survival. Selected patients with histologic transformation, particularly those whose transformation occurs early in the course of their disease and who remain chemosensitive, may experience prolonged survival after autoBMT.     

Histopathology of bone marrow reconstitution after allogeneic bone marrow transplantation

In order to study haematopoietic reconstitution in allogeneic bone marrow transplantation we investigated bone marrow histology in 61 biopsies of 37 patients, treated with HLA-compatible bone marrow grafts for leukaemia or severe aplastic anaemia. The biopsies were taken from the day of transplantation until 100 d after transplantation. Stromal changes, in particular oedema, fibrosis and granulomas, were found during the whole period of observation. These changes were more prominent in biopsies from leukaemia patients than from patients with aplastic anaemia. The cellularity in the biopsies increased until 28 d after bone marrow transplantation and was stable thereafter. Initially, only clusters of cells belonging to a single cell lineage were seen, suggesting that the first outgrowth of haematopoietic cells is by proliferation of committed precursor cells. Long-lasting abnormalities in localization of haematopoietic cells in the bone marrow space and of the myeloid: erythroid ratio were seen; dyserythropoiesis was common.     

The transient appearance of small blastoid cells in the marrow after bone marrow transplantation.

Of 14 patients who underwent allogeneic or syngeneic bone marrow transplantation, 6 had a transient appearance of small blastoid cells in the bone marrow after transplantation. Most of these patients (11) had leukemia, although 3 had severe aplastic anemia. The cells were 8-18 micron in diameter and had scant cytoplasm and dense nuclei with smooth, homogeneous chromatin. They often had distinct nuclear clefts. These cells constituted 4.0-21.3% of the total number of bone marrow cells. They were not reactive with peroxidase, alpha-naphtyl butylate esterase, naphthol AS-D chloroacetate esterase, or periodic acid-Schiff stains. Immunocytochemical analysis revealed that the small blastoid cells expressed terminal deoxynucleotidyl transferase, Ia-like, CD19, and CD10 antigens and cytoplasmic mu heavy chains, indicating a precursor B-cell phenotype. CD20 antigen was not expressed on these cells. The data suggest that cytoplasmic mu may be expressed earlier than CD20 antigen in the differentiation of B-cell lineage. The morphologic, cytochemical, and immunophenotypic characteristics did not distinguish these nonneoplastic cells distinctly from leukemic lymphoblastic cells. The increase of small blastoid cells was a transient and self-limited phenomenon, in contrast to that of neoplastic blasts. These cells should be recognized as a common component of the bone marrow of marrow transplant recipients. The significance and role of these cells in immune recovery and hematopoiesis remain uncertain.     

Second malignancies after allogeneic hematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (allo-HCT) may prolong life and cure patients suffering from otherwise fatal diseases. However, the growing population of long-term survivors has led to the realization of multiple long-term complications, including the risk of second malignancies. Compared to the autologous setting, allo-HCT carries a much higher risk of posttransplant lymphoproliferative disorder (PTLD), which usually occurs within the first year after allo-HCT and is strongly associated with the Epstein-Barr virus (EBV). Treatment-related myelodysplastic syndromes (tMDS) and second leukemias are extremely rare. Both autologous and allo-HCT carry increased risks for second solid malignancies (SSM). The cumulative incidence of SSM continues to increase in each of the largest studies with as much as 20 years of follow-up, likely related to the long latency of radiation-related SSM. Systematic, prospective monitoring, vigilant screening processes, and well-maintained survivorship clinics and databases are absolute necessities, and should be included in the infrastructure of individual transplant centers and networks, with mandatory periodic reporting of second malignancy incidences. Primary care and transplant physicians alike must be aware of the risk of second malignancies after allo-HCT. Most importantly, guidelines should be developed in regard to screening and prevention of second malignancies, so that physicians can provide state-of-the-art counsel and care for the benefit of our patients.     

Histopathology of the lung after bone marrow transplantation.

The histopathological changes in the lungs of 32 patients who died after bone marrow transplantation for leukaemia have been studied and compared with those found in 21 patients treated by conventional chemotherapy. The transplanted patients exhibited a higher incidence of interstitial pneumonitis, vascular lesions and viral infections, particularly cytomegalovirus (CMV), although bacterial and fungal diseases were commoner in the non-grafted subjects. The pathogenesis of interstitial pneumonitis is discussed with specific reference to the possible roles of irradiation, chemotherapy, viruses and the immunosuppressive drug cyclosporin A. Ten patients died of a syndrome characterised clinically by fever, skin rash, fluid retention, uraemia, low serum albumin concentrations, low central venous pressure and acute pulmonary oedema. These patients exhibited intra-alveolar haemorrhagic fibrinous exudation with or without interstitial changes. The aetiology of this syndrome is not known but it occurs more frequently in recipients of mismatched grafts and evidence is presented suggesting that viruses may play a significant causative role. No lesion was identified that could be directly attributed to Graft-versus-Host disease.