Cell Colony-forming Units Research Articles

Bone Marrow Adipocytes: A Novel Negative Regulator of the Hematopoietic Microenvironment.

In the bone marrow (BM), osteoblasts and endothelium constitute functional niches providing positive or negative signals for hematopoietic stem cell (HSC) self-renewal. In addition to hematopoietic cells, endothelial cells, and osteoblasts, adult BM contains numerous adipocytes. Interestingly, the number of adipocytes correlates inversely with the gross hematopoietic activity of the marrow. Whether adipocytes have a direct effect on hematopoietic progenitors or whether they act as mere space-fillers in this context remains unclear. To determine the potential role of bone marrow adipocytes in hematopoiesis, we induced bone marrow-derived OP9 mesenchymal cells to differentiate into either osteoblastic or adipocytic stroma, and then tested their capacity to serve as surrogate HSC niches during in vitro hematopoietic culture in the absence of exogenous growth factors. We found that the presence of BM-derived adipocytes suppresses the expansion of short-term hematopoietic progenitors by at least two fold, as measured by the number of CD45+ cells expanded, the number of colony forming unit cells (CFU-Cs) and the competitive repopulation units (CRUs) during the first two months post-transplant. As an in vivo correlate, we compared the hematopoietic activity within the BM of the adipocyte-poor thoracic vertebrae and the adipocyte-rich proximal tail vertebrae. Indeed, we found that the percentage of HSCs (ckit+Lin-Sca1+), CMPs, GMPs and MEPs was decreased by two fold in the adipocyte-rich BM as determined phenotypically by FACS and functionally by short-term and long-term competitive repopulation assays. Mechanistically, oligonucleotide expression microarrays and conditioned media experiments on the OP9 co-cultures suggest that the inhibition of the progenitor compartment in adipocyte-rich environments is due to the loss of supportive factors (Notch ligands, N-cadherin, Angiopoietin-1, SCF, BMPs and Wnt5a) in addition to the presence of an active inhibitor. Finally, we found bone marrow adipocytes to accumulate in great numbers upon bone marrow ablation, a process that is hindered in genetically adipocyte-deficient mice. Since early BM transplant survival depends on the rapid accumulation of short term hematopoietic progenitors, whose replication we found hindered in adipocyte-rich BM, we were interested to explore whether the absence of adipocytes in the context of BM transplantation would foster faster recovery of lethally irradiated mice. As predicted, circulating leukocyte counts on the third week post-transplant were 3–5 times higher on the recovering fatless mice. Accordingly, the percentage of CFU-Cs, HSCs, CMPs, GMPs and MEPs on day 17 post-transplant was doubled on the adipocyte-depleted mice as compared to their wildtype littermates. We therefore conclude that, as seen in vitro, the presence of adipocytes in the recovering HSC niche is detrimental to the rapid hematopoietic expansion required to reconstitute blood production. We are currently examining the pharmacologic modulation of adipocyte formation for its effects in BM transplantation.

6591 POSTER Analysis of the progostic value of the quantification of plasmatic epidermal growth factor receptor (EGFR) in advanced non-small cell lung cancer (NSCLC) patients

Endothelial progenitor cells (EPCs) are known to play a beneficial role by promoting postnatal vasculogenesis in pathological events, such as ischemic heart disease and peripheral artery disease. However, little is known about the potential of EPCs to restore heart damage tissue. We compared the cardiac differentiation capacity of EPCs isolated from peripheral blood of patients with acute myocardial infarction (AMI) with EPCs obtained from umbilical cord blood (UCB).EPCs from both origins were isolated by density gradient centrifugation and characterized through the use of endothelial markers (UEA-1lectin, CD133 and KDR) and endothelial cell colony-forming unit assay. Cardiac differentiation capacity of EPCs was assessed by immunofluorescence and reverse transcriptase–polymerase chain reaction after 5-azacytidine (5-aza) induction.No significant differences were observed between the number of endothelial cell colony-forming units in peripheral blood of patients with AMI and samples from UCB. Moreover, 5-aza induced the appearance of myotube-like structures and the positive expression of sarcomeric α-actinin, cardiac troponin I and T and desmin in a similar pattern for both cell sources, which indicates a comparable acquisition of a cardiac-like phenotype.For the first time, we have compared, in vitro, the cardiomyogenic potential of EPCs derived from patients with AMI with UCB-derived EPCs. Our data indicate that EPCs obtained from both origins have similar plasticity and functions and suggest a potential therapeutic efficacy in cardiac cell therapy.

Effects of granulocyte colony stimulating-factor in a rat model of acute liver injury

Background/aim Controversial experimental observations suggest that granulocyte colony stimulating-factor may promote hepatic regeneration after hepatectomy and chemical injury either by directly stimulating adult liver cells or facilitating the mobilization of bone marrow cells and their homing to the liver. We investigated whether different schedules of granulocyte colony stimulating-factor administration protect against experimental acute liver injury. Methods Acute liver injury was induced in Sprague-Dawley fed rats by injecting a single intraperitoneal dose of carbon tetrachloride. Recombinant human granulocyte colony stimulating-factor or vehicle was given daily after intoxication (4 days) or before (7 days) and after carbon tetrachloride administration. Liver injury and regeneration were assessed 2 and 4 days after damage. Bone marrow cells mobilization was evaluated by the white blood cell count and the assessment of circulating clonogenic haematopoietic progenitors (colony forming unit-cells). Results In this experimental model, although granulocyte colony stimulating-factor induced the significant mobilization of colony forming unit-cells, the study cytokine had no effect on liver injury (serum alanine amino transaminase level and necrotic index) and liver regeneration (mitotic index and bromodeoxyuridine incorporation), regardless of the administration schedule. Conclusions This study does not support the conclusion that: (1) granulocyte colony stimulating-factor exerts a protective effect against toxic-induced, non-lethal acute liver injury and (2) promotes hepatocyte regeneration.

Negative selection of hematopoietic progenitor cells by continuous magnetophoresis

To evaluate a negative selection technique for the hematopoietic progenitor cell enrichment from clinical leukapheresis product using continuous magnetophoresis. The leukapheresis product was labeled with a tetrameric antibody cocktail (TAC) and magnetic colloid against nonprogenitor leukocytes (StemSep enrichment cocktail kit, Stem Cell Technologies, Vancouver, Canada). The separation of hematopoietic progenitor cells was performed by flow-through magnetophoresis in an annular channel placed coaxially inside a quadrupole magnetic field, in a split-flow thin-cell fractionation configuration (referred to as quadrupole magnetic flow sorter, QMS). The TAC antibody cocktail and the magnetic colloid were titrated to determine minimum effective antibody and magnetic reagent concentrations by measuring cell magnetophoretic mobility (m) distribution using cell tracking velocimetry. Leukapheresis products from eight donors having initial CD34+ cell purity between 0.37 and 9.7% were enriched to the final purity of 30 to 85% and yield of 49 to 84% with a maximum throughput of 6.7 x 10(4) cells/s. The progenitor cell enrichment was accompanied by a more than 3.5 log(10) T-lymphocyte depletion, a significant factor considering the intended application to allogeneic transplantation. Cell colony-forming unit assays showed that there was no deterioration of progenitor cell proliferation and differentiation following the QMS enrichment process. The negative selection method of hematopoietic progenitor cells by continuous magnetophoresis is a promising approach to a process scale-up, important for clinical applications.

Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals

AbstractThe limited vessel-forming capacity of infused endothelial progenitor cells (EPCs) into patients with cardiovascular dysfunction may be related to a misunderstanding of the biologic potential of the cells. EPCs are generally identified by cell surface antigen expression or counting in a commercially available kit that identifies “endothelial cell colony-forming units” (CFU-ECs). However, the origin, proliferative potential, and differentiation capacity of CFU-ECs is controversial. In contrast, other EPCs with blood vessel-forming ability, termed endothelial colony-forming cells (ECFCs), have been isolated from human peripheral blood. We compared the function of CFU-ECs and ECFCs and determined that CFU-ECs are derived from the hematopoietic system using progenitor assays, and analysis of donor cells from polycythemia vera patients harboring a Janus kinase 2 V617F mutation in hematopoietic stem cell clones. Further, CFU-ECs possess myeloid progenitor cell activity, differentiate into phagocytic macrophages, and fail to form perfused vessels in vivo. In contrast, ECFCs are clonally distinct from CFU-ECs, display robust proliferative potential, and form perfused vessels in vivo. Thus, these studies establish that CFU-ECs are not EPCs and the role of these cells in angiogenesis must be re-examined prior to further clinical trials, whereas ECFCs may serve as a potential therapy for vascular regeneration.

Mobilization of Bone Marrow‐Derived Hematopoietic and Endothelial Stem Cells After Orthotopic Liver Transplantation and Liver Resection

In animals, the bone marrow (BM) is a source of liver-repopulating cells with therapeutic potential in case of tissue damage. However, the early response of human BM-derived stem cells (SC) to liver injury is still unknown. Here, we studied 24 patients undergoing orthotopic liver transplantation (OLT) for end-stage liver disease or hepatocellularcarcinoma, and 13 patients submitted to liver resection. The concentration of circulating BM-derived SC was determined by phenotypic analysis and clonogenic assays. Moreover, we assessed the serum level of inflammatory and tissue-specific cytokines. Reverse transcriptase-polymerase chain reaction and fluorescence-in situ hybridization were also used to characterize mobilized SC. At baseline, patients showed a significant lower concentration of circulating CD133(+), CD34(+) SC and clonogenic progenitors (colony-forming unit cells) than healthy controls. However, the time-course evaluation of peripheral blood cells after OLT demonstrated the significant early mobilization of multiple subsets of hematopoietic and endothelial stem/progenitor cells. Cytogenetic and molecular analyses of CD34(+) cells showed the host origin of mobilized SC and the expression of transcripts for GATA-4, cytokeratin 19, and alpha-fetoprotein hepatocyte markers. In contrast with OLT, only total circulating CD34(+) cells significantly increased after liver resection. Mobilization of BM cells after OLT or liver surgery was associated with increased serum levels of granulocyte-colony stimulating factor, interleukin-6, stem cell factor, hepatocyte growth factor, and vascular endothelial growth factor. In summary, we demonstrate that tissue damage after OLT and liver resection induces increased serum levels of multiple cytokines but only ischemia/reperfusion injury associated with OLT results in the remarkable mobilization of BM stem/progenitor cells.

The SCL relative LYL-1 is required for fetal and adult hematopoietic stem cell function and B-cell differentiation

AbstractHematopoietic stem cells (HSCs) arise, self-renew, or give rise to all hematopoietic lineages through the effects of transcription factors activated by signaling cascades. Lyl-1 encodes a transcription factor containing a basic helix-hoop-helix (bHLH) motif closely related to scl/tal, which controls numerous decisions in embryonic and adult hematopoiesis. We report here that Lyl-1 null mice are viable and display normal blood cell counts, except for a reduced number of B cells resulting from a partial block after the pro-B stage. Nevertheless, the deletion of Lyl-1 results in a diminution in the frequency of immature progenitors (Lin–, CD34–, sca-1+, c-kit+ [LSK], and LSK-side population [LSK-SP]) and in S12 colony-forming unit (CFU-S12) and long-term culture-initiating cell (LTC-IC) content in embryonic day 14 fetal liver (E14 FL) and adult bone marrow (BM). More important, Lyl-1–/– E14 FL cells and BM are severely impaired in their competitive reconstituting abilities, especially with respect to B and T lineage reconstitution. Thus, ablation of Lyl-1 quantitatively and functionally affects HSCs, a cell population that transcribes Lyl-1 more actively than their differentiated progenies. Our results demonstrate for the first time that Lyl-1 functions are important for HSC properties and B-cell differentiation and that they are largely distinct from scl functions.

Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells

One factor limiting the therapeutic efficacy of cord blood (CB) hematopoietic progenitor cell (HPC) transplantation is the low cell dose of the graft. This is associated with an increased incidence of delayed or failed engraftment. Cell dose can be increased and the efficacy of CB transplantation potentially improved, by ex vivo CB expansion before transplantation. Two ex vivo CB expansion techniques were compared: (1) CD133+ selection followed by ex vivo liquid culture and (2) co-culture of unmanipulated CB with bone-marrow-derived mesenchymal stem cells (MSCs). Ex vivo culture was performed in medium supplemented with granulocyte colony-stimulating factor, stem cell factor and either thrombopoietin or megakaryocyte growth and differentiation factor. Expansion was followed by measuring total nucleated cell (TNC), CD133+ and CD34+ cell, colony-forming unit and cobblestone area-forming cell output. When compared to liquid culture, CB-MSC co-culture (i) required less cell manipulation resulting in less initial HPC loss and (ii) markedly improved TNC and HPC output. CB-MSC co-culture therefore holds promise for improving engraftment kinetics in CB transplant recipients.

Chemokine expression in human erythroid leukemia cell line AS-E2: Macrophage inflammatory protein-3α/CCL20 is induced by inflammatory cytokines

Normal and malignant hematopoietic cells are shown to express and secrete various cytokines and chemokines, some of which are believed to play an important role in normal and abnormal hematopoiesis in an autocrine/paracrine manner. To explore the possibility of a cytokine/chemokine network participating in the pathophysiology of anemic disorders, we evaluated the ability of inflammatory cytokines to induce chemokine expression using erythroid progenitor cells. Erythropoietin-dependent human leukemia cell line AS-E2 was used as a model of erythroid colony-forming unit (CFU-E) cells. The expression of mRNA of 8 chemokines was examined using RT-PCR, before and after TNF-alpha, IFN-gamma, and IL-1beta stimulation. For MIP-3alpha, the promoter activity was analyzed by luciferase assay and secretion was confirmed by ELISA. The expression of CCR6, the specific receptor for MIP-3alpha, was analyzed by RT-PCR and flow cytometry. Unstimulated AS-E2 cells constitutively expressed transcripts for MCP-4, IP-10, PF-4, IL-8, and MIP-3alpha. Stimulation with TNF-alpha, IFN-gamma, and IL-1beta upregulated MIP-3alpha mRNA expression and induced its protein secretion. Luciferase assay revealed that these cytokines could upregulate promoter activity of the MIP-3alpha gene, possibly through the NF-kappaB pathway. CCR6 mRNA was detected and its intracellular expression was confirmed. These data suggest that inflammatory cytokine-stimulated erythroid progenitors secrete MIP-3alpha, which may function in an autocrine/paracrine manner. Furthermore, the existence of intracellular CCR6 suggests the involvement in cytokine signaling of a MIP-3alpha-dependent internal autocrine mechanism. These mechanisms may play a role in pathophysiology of anemic disorders, such as secondary anemia and bone marrow failure syndromes.

Administration of Low-Dose Cyclophosphamide Reduces Progression of Atherosclerosis in Mice by Controlling the Cellular Microenvironment.

Atherosclerosis develops as a result of multiple inflammatory-fibroproliferative responses and is the primary cause of heart disease and stroke in Western countries. Circulating BM-derived progenitor cells (CFU-Cs or EPCs) can regenerate injured vasculature by accelerating reendothelialization and limiting atherosclerotic lesion formation. Risk factors for coronary artery disease like age and diabetes reduce the number and functional activity of these cells, thus limiting the regenerative capacity. The impairment of stem/progenitor cells by risk factors may contribute to atherosclerotic disease progression. High leukocyte counts, and especially neutrophils, a marker of inflammation have been shown to be an independent risk factor and prognostic indicator of future cardiovascular outcomes. Cyclophosphamide (CY) is a synthetic antineoplastic drug, which reduces white blood cell counts (WBC), especially neutrophils, and under certain circumstances can promote hematopoietic progenitor (colony forming unit-cells, CFU-C) mobilization from the bone marrow (BM) into the circulation.We hypothesized that administration of CY limits atherosclerotic lesion formation by decreasing inflammation-associated cells, and by increasing circulating BM-derived progenitors cells.Apolipoprotein E knockout (ApoE−/ −) mice were fed a high cholesterol diet and received different doses of CY in their drinking water (37.5mg/kg/day, 18.75mg/kg/day, 9.375mg/kg/day or nothing). To control for possible effects on progenitor release a control experiment was set up in which C57/B6 received the highest CY in their drinking water (37.5mg/kg/day) or remained untreated. Peripheral blood was drawn from mice every other week. Peripheral blood mononuclear cells (PBMCs) were isolated and subjected to cultures to detect EPCs and CFU-Cs. In CY-treated mice, WBC and neutrophil counts decreased within the first 10 days after the start of the treatment as compared to non-treated controls, and stayed low over the course of the experiment (until day 70). When apoE mice receiving a high-cholesterol diet were treated with CY, the number of circulating CFU-Cs doubled, whereas no major change was found in the c57Bl/6 control group receiving only Cy. EPCs were not detectable at any time point in the CY or control apoE group. Aortic atherosclerotic tree lesion areas were approximately 50% smaller in apoE-KO mice receiving CY in their drinking water after 12 weeks on a high-cholesterol diet than control animals. Most strikingly, apoE-KO mice treated with CY showed a prolonged survival rate as compared to untreated controls. The presence of macrophage-derived foam cells is a hallmark of the atherosclerotic lesion, and an increase in their content promotes plaque instability.Immunohistochemical analysis revealed that the number of macrophages was reduced in plaques of CY-treated animals, indicating that plaques in these might be more stable. Vascular endothelial growth factor (VEGF) is expressed in atherosclerotic plaques from local macrophages and can induce metalloproteinase-9 (MMP-9). Plasma VEGF levels were lower in the CY-treatment group, coinciding with a decrease in MMP-9 plasma levels.This study demonstrates the potential clinical use of a myelosuppressive agent for the treatment of a benign, but not less “malignant” deadly disease like atherosclerosis.

Haemotoxicity of chlorambucil in the Wistar Hanover rat with particular reference to bone marrow culture, marrow cell apoptosis and levels of FLT3 ligand

We have recently developed a new model of drug-induced chronic bone marrow aplasia (CBMA) in the mouse, which shows features of aplastic anaemia (AA) in humans. Using a regimen of repeated doses of busulphan (BU) we induced late-stage (i.e. chronic) bone marrow depression. There are reports that indicate that other antineoplastic agents [e.g. chlorambucil (CHB), mitomycin, melphalan] may also cause CBMA in the mouse. Wishing to develop a model of CBMA in the rat, we investigated the potential of CHB to induce this change. Female Wistar Hanover (Wistar Han) rats were dosed with CHB (6.3 mg/kg intraperitoneally) on six occasions over 18 days. Animals (n=6–8) were killed and sampled on nine occasions (at 1, 3, 9, 16, 24, 38, 45, 59 and 65 days) after the final CHB dose. A full blood count was performed, and serum was prepared for FLT3 analysis; marrow smears were produced, and the spleen and sternum were placed in histological fixative; femoral marrow suspensions were prepared for assessment of the nucleated cell count [femoral nucleated cell count (FNCC)], levels of apoptosis, and the clonogenic potential of the marrow [colony forming unit cells (CFU-Cs)]. Our results showed that at days 1 and 3 post-dosing, in general, red blood cells (RBCs), lymphocytes and FNCC were significantly reduced in CHB-treated animals; reticulocytes were increased, and platelets and neutrophils were unaffected. At 9 days, parameters in CHB rats were returning to normal, but lymphocytes were still decreased. At 16 and 24 days, many blood parameters were normal, except for reduced lymphocyte counts; this pattern generally remained until the end of the study (day 65). Levels of apoptosis in marrow cells of CHB-treated rats were increased immediately post-dosing, and this elevation persisted until day 16; thereafter, levels were generally normal. Serum FLT3 ligand (FL) levels showed some evidence of increases in CHB rats after dosing. CFU-Cs/femur were significantly reduced in CHB animals after dosing, returning to normal values at day 24. In general, marrow smears showed reductions in the myeloid, erythroid and lymphoid lineages of CHB animals at days 1, 3 and 9, returning to normal at 16 and 24 days. Histology of the spleen showed severe depletion of the white pulp immediately post-dosing in CHB-treated animals. Therefore, it is concluded from these findings that CHB does not induce late-stage (i.e. chronic) bone marrow aplasia in the female Wistar Han rat.

Gene silencing by lentivirus-mediated delivery of siRNA in human CD34+ cells

AbstractTo derive an efficient system for gene silencing in human hematopoietic stem cells (HSCs) we modified a lentiviral vector for small interfering RNA (siRNA) delivery. For this purpose, an H1 promoter-driven siRNA expression cassette was introduced into a lentiviral vector, and the p53 mRNA was chosen as a target for siRNA-mediated gene silencing. Using the recombinant lentivirus we infected human cord blood-derived CD34+ cells and obtained a transfection efficiency of up to 50%, as determined by expression of enhanced green fluorescent protein (EGFP). In EGFP-positive long-term culture-initiating cell (LTC-IC)- and colony-forming unit cell (CFU-C)-derived cells, we observed a reduction of p53 mRNA of up to 95%. Importantly, this reduction remained stable during several weeks of cell culture. Furthermore, p53 gene silencing resulted in decreased p21 mRNA levels and reduced the sensitivity of CD34+ cells toward the cytotoxic drug etoposide. Thus, lentiviral delivery of siRNA can allow for efficient and stable gene silencing in human HSCs and will be very valuable for further gene function studies. (Blood. 2004;103:4511-4513)

Rac it up!

The Rho guanosine triphosphatases (GTPases), including Rho, Rac, and Cdc42, are a family of molecules involved in regulating cellular function, but the specific members critically involved in hematopoeitic cells have not been established. In this report, the investigators generated mice that were conditionally deficient in Rac1, Rac2, or both Rac1 and Rac2 (only in hematopoeitic cells) and focused on the function of blood cells. The absence of Rac1 was associated with an impressive reduction in the ability of hematopoietic stem cell/progenitor (HSC/Ps) to engraft immunodeficient mice. In the absence of both Rac1 and Rac2 there was a massive mobilization of progenitor colony-forming unit cells into the circulation by a mechanism likely dependent on impaired integrin-mediated stem cell adhesion. Both molecules were found to contribute to HSC/Ps growth and migration to stromal-derived factor 1. Rac1 was found to have a relatively larger contribution in regulating cell cycle progression, whereas Rac2 was found to preferentially regulate HSC/Ps apoptosis and actin assembly (cytoskeleton rearrangement). Finally, the authors generated neutrophils from HSC/Ps and demonstrated that these mature cells had similar dependence on both Racs, including a cooperative role in regulating oxidase activity. Collectively, these elegant investigations demonstrate that Rac1 and Rac2 regulate unique aspects of hematopoieitic development and function. The studies provide a mechanistic basis to explain a potential diverse set of immunodeficiency syndromes caused by Rac mutations in patients. Indeed, Rac2 mutations have already been associated with recurrent bacterial infections in select patients. (Gu Y et al. Science 2003;302:445-9.)

Survivin regulates hematopoietic progenitor cell proliferation through p21WAF1/Cip1-dependent and -independent pathways

AbstractThe cyclin-dependent kinase inhibitor p21WAF1/Cip1 and Survivin enhance granulocyte macrophage colony-forming unit (CFU-GM) cell cycle and proliferation and have been implicated as antiapoptotic proteins. We investigated the relationships between p21 and Survivin in primary CFU-GM and c-kit+, lineage-negative (Lin–) cells and demonstrate p21-dependent and -independent pathways whereby Survivin regulates progenitor cell proliferation. Ectopic Survivin enhanced p21+/+ CFU-GM formation and expansion of c-kit+, Lin– cells, whereas p21 gene loss abrogated these effects, indicating a p21 requirement. A dominant-negative form of Survivin and p21 gene deletion accelerated the loss of CFU-GM upon growth factor deprivation, and wild-type Survivin overexpression inhibited apoptosis of p21+/+ CFU-GM and c-kit+, Lin– cells but not p21–/– cells, suggesting that both Survivin and p21 block apoptosis of progenitors and that Survivin-mediated antiapoptosis requires p21. In contrast to the p21-dependent antiapoptotic effects, Survivin increased the proportion of CFU-GM in S-phase in both p21+/+ and p21–/– cells. Furthermore, modulating Survivin expression increased polyploidy in c-kit+, Lin– cells, which was accentuated by p21 deficiency. These results suggest that the Survivin-p21 axis plays an important role in the proliferation of normal hematopoietic cells and that Survivin regulates apoptosis through a p21 WAF1/Cip1-dependent pathway but may control S-phase entry independent of p21.

CD34 +CDw90(Thy-1) + subset colocated with mesenchymal progenitors in human normal bone marrow hematon units is enriched in colony-forming unit megakaryocytes and long-term culture-initiating cells

Objective The progress made in the supportive care of allografts and the identification of mesenchymal stem cells in adult human bone marrow (BM) has prompted renewed interest in the use of BM as a form of cell therapy. With the aim of optimizing the collection of BM cells, we evaluated the hematopoietic and mesenchymal immature cell contents of BM hematon units (HUs), which usually are eliminated during graft processing. Materials and methods Hematopoietic CD34 + progenitors from HU and buffy coat (BC) compartments were characterized in short-term culture. The sorted CD34 +CDw90(Thy-1) + primitive subset was assessed in colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, then further characterized by the expression of additional antigens. In parallel, we evaluated the colony-forming unit fibroblast (CFU-F) number and phenotyped the fresh adherent (D1-3) cells. Results The plating efficiencies of CD34 + cells derived from HU and BC were identical. However, the HU CD34 +CDw90(Thy-1) + subset was enriched in colony-forming unit megakaryocyte (2.3×), LTC-IC (4.6×), and cells coexpressing CD105 (5×). We found a higher frequency of CFU-F (4.7×), considered to be the mesenchymal stem cell-containing population, correlated with an enrichment in fresh adherent (CD45/GPA) −CD14 − cells. Conclusions We show for the first time that functional properties of the CD34 +CDw90 + subset are related to its in vivo location in HU, which may represent the BM mesenchymal reserve compartment. The location in HU of 35.6%, 59.1%, and 58.7% of CD34 + cells, CD34 +CDw90 + LTC-IC, and CFU-F, respectively, justifies the development of a procedure to collect them in order to reduce the therapeutic BM volume.

Autologous transplantation of granulocyte colony-stimulating factor–primed bone marrow is effective in supporting myeloablative chemotherapy in patients with hematologic malignancies and poor peripheral blood stem cell mobilization

AbstractWe assessed the hematopoietic recovery and transplantation-related mortality (TRM) of patients who had failed peripheral blood stem cell mobilization and subsequently received high-dose chemotherapy supported by granulocyte colony-stimulating factor (G-CSF)–primed bone marrow (BM). Studied were 86 heavily pretreated consecutive patients with acute leukemia (n = 21), refractory/relapsed non-Hodgkin lymphoma (n = 41) and Hodgkin disease (n = 17), and multiple myeloma (n = 7). There were 78 patients who showed insufficient mobilization of CD34+ cells (< 10 cells/μL), whereas 8 patients collected less than 1 × 106 CD34+ cells/kg. BM was primed in vivo for 3 days with 15 to 16 μg/kg of subcutaneous G-CSF. Median numbers of nucleated cells, colony-forming unit cells (CFU-Cs), and CD34+ cells per kilogram harvested were 3.5 × 108, 3.72 × 104, and 0.82 × 106, respectively. Following myeloablative chemotherapy, median times to achieve a granulocyte count higher than 0.5 × 109/L and an unsupported platelet count higher than 20 and 50 × 109/L were 13 (range, 8-24), 15 (range, 12-75), and 22 (range, 12-180) days, respectively, for lymphoma/myeloma patients and 23 (range, 13-53), 52 (range, 40-120), and 90 (range, 46-207) days, respectively, for leukemia patients. Median times to hospital discharge after transplantation were 17 (range, 12-40) and 27 (range, 14-39) days for lymphoma/myeloma and acute leukemia patients, respectively. TRM was 4.6%, whereas 15 patients died of disease. G-CSF–primed BM induces effective multilineage hematopoietic recovery after high-dose chemotherapy and can be safely used in patients with poor stem cell mobilization.

Stem cell activity of porcine c-kit+ hematopoietic cells.

A marker for hematopoietic stem cells (HSCs) of pigs, which are considered to be the most suitable donors for clinical xenotransplantation, has not yet been identified. In this study, we examined the HSC activity of porcine c-kit+ bone marrow cells (BMCs). The HSC activity of porcine c-kit+ BMCs was evaluated both in vitro using colony-forming unit (CFU) and cobblestone area-forming cell (CAFC) assays and in vivo in nonobese diabetic/severe combined immunodeficiency transgenic (NOD/SCID-Tg) mice carrying porcine cytokine transgenes. Purified c-kit+ BMCs were substantially enriched for both CFUs and CAFCs in vitro and their transplantation led to long-term porcine hematopoiesis in vivo in mice. Although porcine chimerism was detectable in the peripheral blood of NOD/SCID-Tg mice receiving porcine c-kit- BMCs at early time points after transplantation, the levels were markedly lower than those in mice receiving purified c-kit+ BMCs (0.2%+/-0.14% vs 7.7%+/-1.6% and 0.17%+/-0.17% vs 5.6%+/-2.1% at weeks 3 and 6, respectively). Importantly, all mouse recipients of porcine c-kit+ BMCs showed durable multilineage chimerism (>19 weeks), whereas no recipients of porcine c-kit- BMCs sustained long-term engraftment. Moreover, porcine HSCs that had engrafted for 19 weeks in the recipients of porcine c-kit+ BMCs gave rise to clonogenic progenitors in vitro and reconstituted porcine hematopoiesis in secondary recipients. The present study demonstrates that c-kit is an essential marker of both long-term-repopulating HSCs and progenitor cells with early engraftment capacity.

Clonal analysis of granulocyte-monocyte colony-forming unit cells with the human androgen receptor gene in chronic myeloid leukemia.

Coexistence of Philadelphia chromosome-negative (Ph-) progenitors with the Ph+ clone in the early chronic phase of chronic myeloid leukemia (CML) has been documented in previous reports. A different evaluation of methods is needed to justify the clonality of the residual Ph- progenitors. Therefore, the X chromosome inactivation patterns in individual granulocyte-monocyte colony-forming unit (CFU-GM) colonies were studied with the clonality assay for the human androgen receptor gene. A prerequisite for this evaluation was the validation of T-lymphocytes and buccal cells as control cells representing the constitutional lyonization. The percentages of polyclonal CFU-GM cells were determined in 9 Ph+ women with CML and in 5 healthy women. Results of the clonal analysis of CFU-GM colonies were compared with those from reverse transcriptase-polymerase chain reaction analysis of single colonies for BCR/ABL transcripts. Both methods of CFU-GM cell analysis were in agreement regarding the presence of variable proportions (0%-94%) of normal cells in CML. Our results suggest that (a) T-cells and buccal cells have potential for use as controls for the clonal analysis of CML cases and (b) this method can evaluate the frequency of polyclonal/clonal CFU-GM cells in CML cases and is applicable to the analysis of myeloid clonal disorders that lack specific molecular markers.

Microbial succession in the rhizosphere of live and decomposing barley roots as affected by the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil

The protocol used in the present study was a long-term mesocosm experiment where the microbial succession around live barley roots and subsequent decomposing roots was assessed after seed coating with either the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil. Four diversity measures were used: community level physiological profiles (CLPP), Bacteria-specific polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), actinomycete-specific PCR-DGGE and phospholipid fatty acid (PLFA), as well as total cell counts, colony-forming units (CFU) and culturable spore formers, and spore counts of the Bacillus cereus group. Analysis of non-treated plants provided a baseline description of the natural microbial succession from which effects of the treatments could be evaluated. A microbial succession occurred both in the rhizosphere and around decomposing roots, shown with all three diversity measures. A clear response to root death was found, and a clear distinction between root tip and root base samples. Using the recommended concentration of imazalil and a realistic number of DR54-BN14 for seed coating, transient, initial effects of both treatments on the microbial communities were observed at the root base with the PLFA analysis only. The lack of lasting significant side effects of DR54-BN14 is in agreement with an initial fast reduction in culturable DR54-BN14.

Microbial succession in the rhizosphere of live and decomposing barley roots as affected by the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil

Abstract The protocol used in the present study was a long-term mesocosm experiment where the microbial succession around live barley roots and subsequent decomposing roots was assessed after seed coating with either the antagonistic strain Pseudomonas fluorescens DR54-BN14 or the fungicide imazalil. Four diversity measures were used: community level physiological profiles (CLPP), Bacteria-specific polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), actinomycete-specific PCR-DGGE and phospholipid fatty acid (PLFA), as well as total cell counts, colony-forming units (CFU) and culturable spore formers, and spore counts of the Bacillus cereus group. Analysis of non-treated plants provided a baseline description of the natural microbial succession from which effects of the treatments could be evaluated. A microbial succession occurred both in the rhizosphere and around decomposing roots, shown with all three diversity measures. A clear response to root death was found, and a clear distinction between root tip and root base samples. Using the recommended concentration of imazalil and a realistic number of DR54-BN14 for seed coating, transient, initial effects of both treatments on the microbial communities were observed at the root base with the PLFA analysis only. The lack of lasting significant side effects of DR54-BN14 is in agreement with an initial fast reduction in culturable DR54-BN14.