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Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N=100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean=30.7, SEM=2) decreased and IL-6 levels (mean=4.4, SEM=1) increased with age as did marrow fat (mean=1.2mmfat/g, SEM=0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Vascular remodeling in idiopathic pulmonary arterial hypertension (IPAH) involves hyperproliferative and apoptosis-resistant pulmonary artery endothelial cells. In this study, we evaluated the relative contribution of bone marrow-derived proangiogenic precursors and tissue-resident endothelial progenitors to vascular remodeling in IPAH. Levels of circulating CD34+ CD133+ bone marrow-derived proangiogenic precursors were higher in peripheral blood from IPAH patients than in healthy controls and correlated with pulmonary artery pressure, whereas levels of resident endothelial progenitors in IPAH pulmonary arteries were comparable to those of healthy controls. Colony-forming units of endothelial-like cells (CFU-ECs) derived from CD34+ CD133+ bone marrow precursors of IPAH patients secreted high levels of matrix metalloproteinase-2, had greater affinity for angiogenic tubes, and spontaneously formed disorganized cell clusters that increased in size in the presence of transforming growth factor-beta or bone morphogenetic protein-2. Subcutaneous injection of NOD SCID mice with IPAH CFU-ECs within Matrigel plugs, but not with control CFU-ECs, produced cell clusters in the Matrigel and proliferative lesions in surrounding murine tissues. Thus, mobilization of high levels of proliferative bone marrow-derived proangiogenic precursors is a characteristic of IPAH and may participate in the pulmonary vascular remodeling process.

The use of mobilized hematopoietic progenitor cells (HPC) has largely replaced the use of bone marrow HPC for autologous and allogeneic transplantation; however, the mechanisms of HPC mobilization remain unclear. A better understanding of these mechanisms, may allow the development of improved (potentially more rapid and/or higher yield) HPC mobilization strategies, especially for patients who mobilize poorly using current mobilization protocols. Clinically, granulocyte colony-stimulating factor (G-CSF) is widely used to induce HPC mobilization, and evidence suggests that metalloproteinase enzymes released by activated granulocytes play an important role in the G-CSF-induced HPC mobilization. These enzymes may act to disrupt putative cell-cell and/or cell-extracellular matrix interactions within the hematopoietic microenvironment thereby releasing HPC into the blood. Matrix metalloproteinase-9 (MMP-9) appears to be important for G-CSF-induced mobilization. Using an MMP-9 knock-out (KO) mouse model, we investigated the role of MMP-9 in G-CSF and erythropoietin (EPO)-based HPC mobilization at clinically relevant cytokine doses. There were few hematologic or hematopoietic differences between the wild-type and MMP-9KO mice during steady-state hematopoiesis. When treated subcutaneously with EPO (500 U/kg per day) and G-CSF (15 microg/kg per day) for 5 days and assayed on day 6, similarly increased extramedullary hematopoiesis and numbers of HPC in the spleen and blood were observed for both the wild-type and MMP-9KO mice. These data demonstrate that MMP-9 is not required for EPO + G-CSF mobilization and that alternative mobilization mechanisms must be active at clinically relevant cytokine concentrations.

Bone marrow (stem/progenitor) cells have been shown to "differentiate" into cells in multiple tissues, including lung. A low number of hematopoietic stem/progenitor cells also circulate in peripheral blood. The physiologic roles of these cells are still uncertain. This study was designed to test, using parabiotic mice that were joined surgically, whether stem/progenitor cells in blood contributed to the regeneration of lung after injury. Parabiotic mice were generated surgically by joining green fluorescent protein transgenic mice and wild-type littermates. These mice developed a common circulation (approximately 50% green cells in blood) by 2 weeks after surgery. The wild-type mouse was either uninjured or lethally irradiated or received intratracheal elastase or the combination of radiation with intratracheal elastase injection. Radiation or the combination of radiation with elastase significantly increased the proportion of bright green cells in the lungs of the wild-type mice. Morphologically, interstitial monocytes/macrophages, subepithelial fibroblast-like interstitial cells, and additionally type I alveolar epithelial cells immunostained for green fluorescent protein in wild-type mice. Approximately 5 to 20% of lung fibroblasts primary cultured from injured wild-type mice were green fluorescent protein expressing cells, indicating their blood derivation. This study demonstrates that stem/progenitor cells in blood contribute to the repair of lung injury in irradiated mice.

Bone marrow (stem) cells can differentiate into cells in multiple tissues, including lung. Conversely, there are reports that cells of nonhematopoietic tissues (brain, muscle) can give rise to lymphohematopoietic cells. Here we show that the lung contains cells capable of giving rise to lymphohematopoietic cells when transplanted to irradiated recipients. Whole lung cell suspensions, lung side population (SP) cells, and CD45(+/-) lung cells obtained from male transgenic enhanced green fluorescent protein-expressing mice were transplanted intravenously to total body irradiated female mice. Green fluorescent cells were recovered from the circulation and phenotyped for their expression of lymphohematopoietic markers (CD3, CD4, CD8, B220, Gr-1, and Mac-1). Lung SP cells were composed of heterogeneous populations and had less ability to give rise to lymphohematopoietic cells than did bone marrow SP cells. Furthermore, the ability of cells from the lung of aged mice to generate lymphohematopoietic progeny was equivalent to that of cells from young mice. Cells from lung with radioprotective and lymphohematopoietic reconstituting abilities were CD45(+). CD45(+) cells in the lung cells have lymphohematopoietic stem/progenitor cell characteristics, and this has implications for cell or gene therapy applications.

Background: Cytokine-induced mobilization of hematopoietic stem/progenitor cells to the circulation facilitates efficient harvest of blood stem cells by leukapheresis. Up to 30% of autologous, and 10-20% of allogeneic blood stem-cell donors respond poorly to mobilizing cytokines and preliminary studies implicated a circulating inhibitor of mobilization., Methods: In this study, plasma from 11 allogeneic and 23 autologous stem cell donors was assayed for inhibition of mobilization in mice., Results: There were significant correlations between CD34(+) cells collected/kg human donor weight and spleen weight, CD34(+) CD45(+) cells, GMCFC and HPP-CFC per spleen in murine recipients of these plasma samples. Overall, there was a positive association between transforming growth factor beta (TGF-beta) levels and CD34(+) cells per liter of blood processed (LBP). However, when arbitrarily segregated into good versus poor mobilizers, based on less or greater than 15 million CD34(+) cells collected per LBP, the majority (64%) of normal donors were good mobilizers. The majority of the poor mobilizers (83%) were patients. For a subset of 12 individuals whose plasma strongly inhibited mobilization in the mouse, a significant positive correlation of the extent of inhibition with TGF-beta levels was found. For 11 individuals whose plasma, based on colony assays, enhanced mobilization when injected into mice, no correlation with TGF-beta levels was evident., Discussion: Elevated plasma TGF-beta levels in some stem-cell donors may be associated with poor stem-cell mobilization. It remains to be determined whether elevation of TGF-beta levels is a cause of, or a compensatory response to, poor mobilization.

This study was designed to examine the relationship of prior therapy, bone marrow metastases, mobilization, and blood progenitor/stem cell (BSC) collection in breast cancer patients. Cells were collected from 19 breast cancer patients during steady state (nonmobilized group) and from 69 breast cancer patients after cytokine administration (mobilized group). Characteristics of the patients were compared with the cells obtained. A significant inverse association was found between the number of chemotherapy regimens the patients had received prior to BSC collection and the mononuclear cell (MNC) count of the product per liter of blood processed (LBP) with apheresis (P = .0006) and the granulocyte monocyte/macrophage colony-forming cell (GM-CFC) numbers per LBP (P = .0002). This association was evident in both mobilized and nonmobilized patients. Similar results were seen in those 25 patients who had received prior radiation therapy (MNC/LBP, P = .0003; GM-CFC/LBP, P = .0004). Patients in both the mobilized and nonmobilized groups with marrow metastases at the time of collection also had significantly lower levels of MNC/LBP (P = .0039) and GM-CFC/LBP (P = .0001) than did those without marrow metastases. The findings suggest that prior administration of radiation therapy and/or chemotherapy and the presence of marrow metastases all negatively impacted the collection of mobilized and nonmobilized progenitor cells from breast cancer patients. The mechanisms of this impact are not understood.

Objective: To determine if circulating factors influence strain-specific responses to administration of hematopoietic stem-cell mobilizing cytokines, a murine model was employed., Methods: Plasma aliquots from intact DBA2, Balb/c, and C57Bl/6 mice were injected into intact Balb/c mice prior to delivery of mobilizing cytokines. Control Balb/c mice were injected with mobilizing cytokines alone. Plasma from hemi-body irradiated Balb/c mice, known to inhibit mobilization, was also injected into Balb/c mice. Twenty-four hours later, spleen cells were harvested and assayed for granulocyte-macrophage colony-forming cells (GM-CFC) and high-proliferative-potential colony-forming cells (HPP-CFC). Simultaneously harvested blood aliquots were assayed for CD45(+)/CD34(+) cells using flow cytometric techniques., Results: Mice receiving plasma from any source demonstrated significant inhibition of mobilization of HPP-CFC and GM-CFC to the spleen as compared to mobilized controls; for HPP-CFC, plasma from C57Bl/6 mice was more inhibitory than plasma from Balb/c (p = 0.001) or from DBA2 mice (p = 0.01), while for GM-CFC, plasma from C57Bl/6 mice was more inhibitory than Balb/c plasma but not more inhibitory than DBA2 plasma. Mice injected with plasma from previously irradiated Balb/c mice exhibited the expected HPP-CFC and GM-CFC mobilization inhibition, which was not statistically different from the inhibition seen in animals that received C57Bl/6 plasma. Mobilization of CD34(+)/CD45(+) cells to the blood also appeared to be inhibited by pretreatment with C57Bl/6 plasma, but not DBA2 plasma., Conclusion: These data suggest that strain-specific patterns of mobilization may be influenced by a circulating mobilization inhibitor(s).

Background: Allogeneic blood stem-cell donors demonstrate more vigorous mobilization of CD34(+) cells to the circulation in response to cytokine administration than do autologous donors. Transforming growth factor (TGF-beta1) has been implicated as a mobilization inhibitor. A study was designed to determine whether plasma TGF-beta1 levels are elevated in cytokine-mobilized autologous cancer donors compared with cytokine-mobilized normal donors., Methods: Plasma collected from 29 autologous cancer donors and 33 normal allogeneic stem-cell donors following administration of mobilizing cytokines just prior to the first collection was assayed for TGF-beta1 using a sandwich-type ELISA. Plasma from three volunteers not treated with cytokine was also analyzed. Comparisons were made using the Student's t test on log-transformed data., Results: Average TGF-beta1 levels in the plasma of cancer patients were significantly higher than in allogeneic stem-cell donors (4.4 ng/mL versus 7.2 ng/mL; p = 0.038). The allogeneic donors required fewer collections to harvest greater numbers of CD34(+) cells and colony-forming unit granulocyte-macrophage (CFU-GM) than autologous donors. Plasma from three untreated volunteers had mean TGF-beta1 levels of 0.36 ng/mL, with all three levels below the 25th percentile for allogeneic donors and the 5th percentile for cancer patients., Discussion: Cytokine administration was associated with increased plasma TGF-beta1 levels. The levels were higher in cancer patients being mobilized for stem-cell collection than in allogeneic blood stem-cell donors. These differences could underlie the increased number of apheresis procedures required to harvest autologous graft products from cancer patients.

Mobilization of stem and progenitor cells into blood, which facilitates the collection of blood-derived autograft and allograft products, can be accomplished with administration of myelosuppressive chemotherapy, hematopoietic growth factors, or both. Autologous donor indifference to mobilization attempts has been correlated with prior administration of chemotherapy and radiation therapy. To investigate whether concurrent administration of radiation therapy inhibits mobilization, five daily injections of a potent combination of mobilizing cytokines, 500 U/kg erythropoietin (EPO) plus 15 microg/kg G-CSF, were administered each morning to Balb/c mice. Each afternoon, a 2 Gy fraction of Co-60 radiation was administered to either the lower limb or the upper or lower hemibody. Each day, mice were necropsied, and blood stem cell mobilization was determined by assaying the number of hematopoietic colony-forming cells in the blood and in the spleen. Unirradiated cytokine-injected mice showed a significant mobilization effect evident as increased colony-forming cells in blood and spleen compared with saline-injected unirradiated controls. The irradiated mice showed markedly inhibited or absent mobilization regardless of the part of the body irradiated. To investigate the mechanism of radiation-induced mobilization inhibition, heparinized plasma was obtained from mice whose lower bodies were irradiated with 2 Gy 18 h previously, and 0.5 ml was injected i.v. into intact mice 10 min before they received 15 microg/kg G-CSF and 500 U/kg EPO. Unlike mice that received G-CSF + EPO only and showed mobilization of progenitors from marrow to spleen, recipients of plasma from irradiated mice before and after cytokine administration showed significantly reduced mobilization of progenitors. Thus, radiation-induced inhibition of stem cell mobilization is mediated by an unidentified circulating factor.

Deliberately increasing the number of hematopoietic stem and progenitor cells in the circulation allows faster and more efficient collection of sufficient cells for transplantation in both the allogeneic and autologous settings. These mobilized stem cells, when transplanted, provide quicker hematopoietic recovery for the patient than do nonmobilized blood stem cells or steady-state marrow-derived stem cells. Currently used clinical procedures to produce stem cell mobilization include administration of G-CSF or GM-CSF, either as single agents or in combination with myelosuppressive chemotherapy. Some autologous blood stem cell donors exhibit indifference to currently applied mobilization therapies. This failure to mobilize has been associated with prior stem cell toxic therapy, e.g., radiation therapy and chemotherapy, but the association is incomplete. The observation that occasional normal donors have failed to respond to mobilization therapy indicates that factors other than stem cell damage could also be involved. Recently, a murine model has provided evidence that a circulating factor inhibits mobilization in some settings. Preliminary investigations have suggested that a circulating factor may inhibit mobilization of human hematopoietic progenitor cells in some instances. Studies to identify this factor(s) are underway. The mechanisms of blood stem cell mobilization are still poorly understood and there continues to be the potential to improve this process.

The maintenance of hematopoietic progenitor cells as assayed in the mixed colony (CFU-GEMM) assay in human long-term bone marrow cultures was compared between normal allogeneic marrow transplantation donor collections and those from candidates for high-dose therapy and autologous bone marrow transplantation (ABMT). To be eligible for ABMT, patients were required to have a histologically normal appearing bone marrow and therefore any tumor contamination was at minimal levels and detectable only after evaluation of the cultured harvests. Marrow from 15 normal donors, 36 patients with breast cancer, and 30 patients with Hodgkin's disease was evaluated. The number of mononuclear cells placed in culture was standardized. In all groups, significantly more progenitor cells were recovered at 4-6 weeks of culture that at 12-14 weeks. At 4-6 and 12-14 weeks, there were no significant differences in the number of progenitor cells recovered from the cultures of normal donors and tumor negative cultures of breast cancer or Hodgkin's disease patients. However, following 4-6 and 12-14 weeks of culture, progenitor cell numbers of cultures which contained breast cancer cells were significantly higher than the pooled values for cultures from the concurrent normal controls, and those from breast cancer and Hodgkin's disease patients with tumor negative cultures. These results suggest that minimal breast cancer cell contamination of the bone marrow can influence the production of marrow progenitor cells. Exposure to prior chemotherapy or radiation therapy does not appear to be the cause of this effect. The most likely mechanism is the local production of cytokines by the tumor cells, although a process involving direct adhesive contact of the tumor cells with hematopoietic cells, which is sometimes observed in semisolid cultures, cannot be excluded.

Purpose: To evaluate the outcomes in 65 consecutive patients with non-Hodgkin's lymphoma (NHL) undergoing high-dose therapy (HDT) and autologous transplantation based on initial marrow involvement and the presence or absence of minimal disease in the hematopoietic harvests., Patients and Methods: Patients with any history of histologic evidence of marrow tumor underwent autologous peripheral-blood stem-cell transplantation (PSCT), whereas others underwent autologous bone marrow transplantation (ABMT). Patients who underwent ABMT were further segregated retrospectively into two groups depending on whether there was evidence by cell culture and/or Southern analysis of minimal tumor in the marrow harvest., Results: Comparable proportions (58% to 60%) of patients in each of the two groups (PSCT and ABMT) achieved a complete clinical remission (CR) at 100 days. For patients who achieve a CR, the actuarial relapse-free survival rate at 5 years for PSCT patients who received a tumor-negative apheresis harvest was 64%, compared with 57% for patients who received a tumor-negative bone marrow harvest and 17% for patients who received a histologically negative but minimally contaminated bone marrow harvest. Lymphoma grade and phenotype were not significant predictors of outcome., Conclusion: The observation that survival was significantly better in the groups of patients who received tumor-negative harvests and worse for patients who received minimally contaminated harvests suggests that tumor cells, even at minimal levels, reinfused in the transplanted harvest are responsible for progression in a proportion of patients who achieve a CR following HDT, although other biologic characteristics of the tumor could also be important. A relatively good outcome can be achieved with HDT and PSCT, even in patients with a significant marrow tumor burden.

We examined phosphorothioate oligodeoxyribonucleotides (ODNs) directed against bcr in exon 3 or exon 2, which are rearranged with exon 2 of abl (B3A2 and B2A2) at t(9;22) of chronic myelogenous leukemia (CML). Since these ODNs are designed to be CML cell specific, we studied their effects on the human CML cell line K562, which is known to have B3A2 rearrangement, and leukemic cells from patients, as well as normal hematopoietic stem cells in vitro. In vitro experiments were performed to determine a potential role of these two ODNs as ex vivo purging agents. Incubation of B3A2 antisense at 40, 80, and 120 micrograms/ml with K562 CML cells for 72 hours at 37 degrees C resulted in 44%, 56%, and 63% reduction of CFU-L as compared to controls. In contrast, B3A2 sense and B2A2 antisense had no significant growth inhibitory effect on K562 cells. Incubation of B3A2 and B2A2 antisense ODNs at concentration of 80 micrograms/ml at 37 degrees C for 36 hours with normal peripheral blood stem/progenitor cells (PBSC) resulted in 124% and 98% CFU-GM formation as compared to untreated controls, respectively. However, incubation of PBSC with B3A2 and B2A2 sense-ODNs resulted in a 22% and 44% reduction in CFU-GM, respectively. In order to determine the ex vivo purging effects of bcr-abl ODNs, the K562 cells were mixed with PBSC from normal donors at a ratio of 1:20 (CML:PBSC). The mixture of cells was then incubated with B3A2 antisense at 80 micrograms/ml for 36 hrs at 37 degrees C. After incubation, no CML cells were detected by fluorescence in situ hybridization (FISH) as compared to untreated controls. These results were confirmed by RT-PCR using bcr-abl primers and mRNA isolated from the mixture of cells. Further, these results support the hypothesis that bcr-abl antisense ODNs are potentially effective agents for ex vivo purging of autologous stem cells before transplantation to eliminate/reduce the burden of leukemic cells. No significant toxicity to normal hematopoietic stem/progenitor cell population by the bcr-abl antisense ODNs was observed. Although unanticipated reductions in normal hematopoietic progenitor cells (CFU-GM) were observed with sense ODNs, no reduction in CFU-GM was observed with unrelated phosphorothioate ODN controls.

A trial to determine the usefulness of recombinant human erythropoietin (rhEpo) as a mobilizing cytokine for patients with previously treated relapsed malignancies was performed. An initial peripheral stem cell apheresis collection was conducted during steady-state hematopoiesis for each patient to provide baseline data. rhEpo, 200 U/kg/day, was administered subcutaneously until the last apheresis procedure was completed. Immediately after the fourth daily dose of Epo, apheresis procedures were resumed and continued beyond five collections, when necessary, to accrue a total of 6.5 x 10(8) mononuclear cells (MNCs)/kg. Eight female and four male patients (median age = 44 years) were evaluated. Five to 14 (median = 8) apheresis procedures were performed for each patient. Toxicity attributable to Epo administration was negligible. Mobilization effects, as determined by an increase in the number of colony-forming units granulocyte/macrophage (CFU-GM) and burst-forming units-erythroid (BFU-E) in the apheresis products after Epo administration, were observed in all patients. Nine patients received high-dose chemotherapy and Epo-mobilized peripheral stem cell transplantation (PSCT). Beginning the day of the transplant, GM-CSF was administered until neutrophil recovery was satisfactory. The median time to recover 0.5 x 10(9)/L granulocytes was 16 days after PSCT. Epo appears to have mobilization properties. Further studies are needed to determine the clinical usefulness of Epo as a mobilizing cytokine. The addition of Epo to other mobilizing cytokines may provide increased effectiveness without adding toxicity.

The OMA-AML-1, acute myelogenous leukemia cell line is unique in that it spontaneously maintains both a CD34+ precursor cell compartment and a CD15+ differentiating cell compartment in vitro. A third transitional cell type with co-expression of CD34 and CD15 can also be identified in in vitro cultures. The cell line shows dynamic fluctuations in the relative sizes of these three cell compartments in suspension culture. In contrast, OMA-AML-1 fails to show phenotypic or morphologic evidence of differentiation when grown subcutaneously in immunodeficient mice. OMA-AML-1 responds to a number of hematopoietic cytokines. Delineation of cytokine responses on FACS isolated populations of CD34+ versus CD15+ cells demonstrated that proliferative responses occurred primarily at the level of the precursor cell (CD34+) while the production of endstage eosinophils occurred within the CD15+ compartment. OMA-AML-1 mimics a number of features of normal hematopoiesis and is proving to be a useful in vitro model for the study of hematopoietic differentiation.

We examined the effect of catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on murine committed megakaryocyte progenitor cells, the megakaryocyte-colony forming unit (CFU-Meg). More mature cells of the megakaryocyte series have the capacity for active uptake of catecholamines, and we speculated that the CFU-Meg would also take up 6-OHDA and be selectively killed. CFU-Meg were much more sensitive to the effects of this agent than were granulocyte-macrophage colony forming units (CFU-GM) or spleen-colony forming units. Co-incubation with catalase, which would destroy hydrogen peroxide generated extracellularly by the autoxidation of 6-OHDA, ablated 6-OHDA toxicity towards CFU-GM, but also significantly reduced the effect on CFU-Meg. Mazindol, a selective dopamine uptake inhibitor did not alter 6-OHDA effect on either CFU-Meg or CFU-GM. Finally, CFU-Meg were no more sensitive to incubation with hydrogen peroxide than were CFU-GM. These data suggest that CFU-Meg, unlike their more mature progeny, do not actively concentrate 6-OHDA, and the excess toxicity of this agent towards CFU-Meg is probably due to increased sensitivity to autoxidation products of 6-OHDA, other than hydrogen peroxide, generated extracellularly.

The role of hematopoietic microenvironments in the regulation of maturation and differentiation of hematopoietic cells, although heavily debated, remains uncertain. Several investigators have suggested that the adherent "stromal" cell populations, which grow as colonies in cultures of lymphomyeloid tissues, include the cells involved in such regulatory processes. Grossly, the colonies described by several investigators appear similar morphologically, and the cells giving rise to them have been variously termed 1) fibroblast colony forming cells (FCFC), 2) plaque forming units-culture (PFU-C), 3) macrophage colonies, and 4) marrow stromal cells. FCFC have been reported to re-establish their parent microenvironment when transplanted in an allogeneic system. In this study, cloned and enriched cell populations obtained from such colonies in cultures of murine lymphomyeloid tissues have been characterized by their growth in culture and using morphological, histochemical, and electron microscopic techniques. The results demonstrated that, although the initial stromal colonies appeared to be identical, the constituent cell types varied considerably. Some colonies were comprised primarily of macrophages, while others appeared to contain predominantly fibroblasts; two additional cell types that established colonies have not yet been satisfactorily identified. These results demonstrate the heterogeneity of lymphomyeloid stromal colonies. There is a need for caution in the analysis of experiments in which uncharacterized stromal cell colonies are transplanted or employed as supporting monolayers in culture systems in experiments designed to evaluate the origins and functions of lymphohematopoietic stroma.

Following the administration of similar doses of nitrogen mustard (4 mg/kg) to different strains of mice, wide variations in the subsequent degree of splenomegaly were observed, implying strain differences in the role of the spleen in the compensatory erythropoietic response to haematopoietic stress. This investigation was undertaken to determine whether or not these differences were related to the size of the haematopoietic stem cell compartment size in the various strains of mice. Groups of 4 different strains of mice (Swiss Webster, A/J, C57BL/6J and CS1/ASH) were injected i.v. with nitrogen mustard (4 mg/kg body weight) and autopsied at regular intervals up to 20 d post-injection. At autopsy, the wet weight of the spleen was determined. Subsequently, groups of the same 4 strains of mice were exposed to single doses of wholebody gamma-irradiation in the range of 500-900 rads. 9 d after gamma-irradiation the mice were autopsied, their spleens removed, and the number of endogenous spleen colonies determined. The greatest degree of splenomegaly was observed in the C57BL/6J mice. The Swiss Webster mice showed no splenomegaly during the time period studied. There existed a linear inverse relationship between the maximum degree of splenomegaly observed and the dose of wholebody gamma-irradiation required to completely eliminate endogenous spleen colonies. This data is in accord with the hypothesis that there exists an inverse relationship between the extent of splenomegaly observed following haematopoietic stress and the haematopoietic stem cell compartment size.

This study evaluated the usefulness of a matrix culture system, the artificial capillary culture system, for the growth of hematopoietic cells, the maintenance of CFUS and the collection of GM-CSF. The system was compared to monolayer, gelfoam sponge and Dexter liquid cultures. Monolayer cultures of adherent lymphohematopoietic stromal cells were prepared from bone fragments, whole bone marrow (Dexter system), spleen, fetal liver and thymus preparations. Morphologically, all the adherent cell populations were similar and tended to accumulate macrophages. Despite these morphological similarities, the ability of bone marrow adherent cell monolayers to maintain CFUS early in the culture period was significantly greater than adherent cells from other sources. Low but significant levels of GM-CSF were detected by bioassay in the supernatants of all but bone marrow adherent cell cultures. However, adherent cells selected from whole marrow cultures by differential trypsinization contained bioassayable levels of CSF suggesting that Dexter cultures contain a supernatant inhibitor/inactivator of GM-CSF. The greatest capability of the artificial capillary culture system was to increase about 500-fold over monolayers the GM-CSF collected from bone fragment derived stromal cells. Unfortunately, CFUS maintenance was poor, the system was more expensive than monolayer cultures and in our hands, suffered many mechanical failures usually resulting in loss of sterility. Histological evaluation suggested there was inadequate matrix for optimal attachment and growth of lymphohematopoietic stromal cells. Even so this system has a much greater potential for development than gelfoam sponges. Overall, Dexter cultures appear to be the most useful system currently available for the study of hematopoiesis and hematopoietic regulatory interactions.

Mobilization of hematopoietic stem cells to the circulation facilitates their collection, thereby providing a non-marrow source of these cells for transplantation. Hematopoietic cytokine administration induces mobilization for most, but not all, donors. Because the underlying biology of mobilization is not well understood, improving the process on a rational basis is difficult. The design of an in vitro mobilization model was pursued to facilitate investigations of the process. MS5 murine stromal cell line cells were grown to confluence on microporous transwell membranes. Murine femoral marrow plugs were placed on top of the prepared transwell membranes. The transwells were then seated in wells containing media and hematopoietic growth factors. Cells that were released from the marrow plugs over time and migrated through the stromal layer into the wells were assayed for stem cell/progenitor cell characteristics. Few or no GM-CSF (progenitors) were found in wells containing media alone or media plus mobilizing cytokines after 24 h. After 120 h, the numbers of cells in the cytokine-containing wells increased, as did the numbers of CD34 + cells. Cells in the wells at the time progenitor cells were most frequent were shown to include side population (SP) hematopoietic stem cells. After 120 h in the presence of cytokines, cells pooled from the wells were transplanted to lethally irradiated mice. Eighty per cent of the transplanted mice survived 30 days or more, demonstrating that radioprotective stem cells were present in the wells. An ex vivo model has been designed that may aid investigations of the various steps of stem cell mobilization. [ABSTRACT FROM AUTHOR]

Background BM cells have been shown to give rise to progeny of various cell lineages, including cells in lung and liver. This investigation evaluated whether purified BM mononuclear cells and side population (SP) cells that have hematopoietic stem-cell activity also had this property; whether a TBI preparative regimen was necessary for engraftment; and where BM-derived cells were engrafted. Methods Either 1-3 million BM mononuclear cells or 2000 BM SP cells from transgenic enhanced green fluorescent protein-expressing (EGFP) mice were transplanted i.v. to unirradiated or 7-9.5 Gy irradiated recipients. Results Flow cytometric analysis showed that lung cells (mean 45%, range 4-70%) and liver cells (mean 4%, range 0.4-8.3%) from irradiated, but not unirradiated recipients, were EGFP donor-derived. Similar results were obtained transplanting BM mononuclear cells or SP cells. Morphologically, donor-derived cells in the lung were primarily monocytes and macrophages. Additionally, lung fibroblasts and Type I, but not Type II, alveolar cells and rare cells in the bronchial epithelium were donor BM derived. In the liver, Kupffer cells, inflammatory cells and small clusters of hepatocytes, but not bile duct cells, were donor-derived. Discussion BM mononuclear and SP cells generated progeny in some compartments of the lung and liver, but only in TBI recipients. Stem cells in BM can contribute to repair of tissue injury in some compartments, but not to the same extent in the lung and liver. [ABSTRACT FROM AUTHOR]

Intentional mobilization of hematopoietic/stem cells into the circulation has improved the efficiency of their collection. Transplantation of mobilized blood stem cells to patients with marrow aplasia results in a faster pace of hematopoietic recovery than transplantation of marrow-derived stem cells. Autologous and allogeneic hematopoietic stem cell transplantation are increasingly performed with blood-derived cells. Donors of both autologous and allogeneic blood stem cells do not always respond well to therapies designed to produce mobilization. Autologous donors may respond poorly as a result of myelotoxic damage inflicted by prior antitumor therapy, but this explanation is not valid for allogeneic donors. The mechanism(s) involved in the process of mobilization are incompletely understood. Until these mechanisms are elucidated, methods to improve mobilization vigor on a rational basis will not be obvious. In the meanwhile, clinical observations may provide some hints regarding the whys and hows of mobilization and permit incremental improvements in this process.Bone Marrow Transplantation (2003) 31, 319-329. doi:10.1038/sj.bmt.1703837 [ABSTRACT FROM AUTHOR]