Cell Colony-forming Units Research Articles

Circulating endothelial progenitor cell levels and function in patients who experienced late coronary stent thrombosis

The pathogenesis of late coronary stent thrombosis may be related to impaired arterial healing. Endothelial progenitor cells (EPCs) have been shown to play an important role in repair and re-endothelialization following vascular injury. We hypothesized that patients who develop late stent thrombosis may have reduced or dysfunctional EPCs, and aimed to compare EPC level and function in patients who experienced stent thrombosis vs. matched controls. Patients who developed late (> 30 days) stent thrombosis within the past 3 years were compared with matched patients who underwent stenting and did not develop stent thrombosis. All patients had blood samples taken ≥ 3 months from the stent thrombosis or index procedure. The proportion of peripheral mononuclear cells (PMNCs) expressing vascular endothelial growth factor receptor 2 (VEGFR-2), CD133, and CD34 was evaluated by flow cytometry. Endothelial progenitor cell colony forming units (CFUs) were grown from PMNCs, characterized and counted following 7 days of culture. The two groups (n = 30 each) were well-matched (93.3% men, mean age 60-62 years, 33.3% diabetes, 73-80% DESs). The proportion of cells co-expressing VEGFR-2, CD133, and CD34 was lower in the stent thrombosis group compared with the control [VEGFR-2(+)CD133(+): 0.18% (0.03-0.41%) vs. 0.47% (0.16-0.66%), P = 0.01; VEGFR-2(+)CD34(+): 0.32% (0.22-0.70%) vs. 0.66% (0.24-1.1%), P = 0.03]. The number of EPC CFUs was also lower in the stent thrombosis group [3.9% (3.2-5.5%) vs. 8.3% (6.5-13.4%) colonies/well, respectively, P < 0.0001]. Patients who suffered late coronary stent thrombosis appear to have reduced levels of circulating EPCs and impaired functional properties of the cells. These findings require validation by further studies, but may contribute to understanding the pathogenesis of late stent thrombosis.

5% Dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO

Cell number and viability are important in cord blood (CB) transplantation. While 10% dimethyl sulfoxide (DMSO) is the standard medium, adding a starch to freezing medium is increasingly utilized as a cytoprotectant for the thawing process. Similar to hetastarch, pentastarch has the advantages of faster renal clearance and less effect on the coagulation system. We compared a lower DMSO concentration (5%) containing pentastarch with 10% DMSO and performed cell viability assay, colony-forming units (CFUs), and transplantation of CB cells in NOD/SCID IL2Rγ(null) mice. CB cells in 5% DMSO/pentastarch had similar CD34+, CD3+, and CD19+ cell percentages after thawing as fresh CB cells. CB cells in 5% DMSO/pentastarch had higher viability (83.3±9.23%) than those frozen in 10% DMSO (75.3±11.0%, p<0.05). We monitored cell viability postthaw every 30 minutes. The mean loss in the first 30 minutes was less in the 5% DMSO/pentastarch group. At the end of 3 hours, the viability decreased by a mean of 7.75% for the 5% DMSO/pentastarch and 17.5% for the 10% DMSO groups. CFUs were similar between the two cryopreserved groups. Frozen CB cells engrafted equally well in IL2Rγ(null) mice compared to fresh CB cells up to 24 weeks, and CB cells frozen in 5% DMSO/pentastarch engrafted better than those in 10% DMSO. Our data indicate that the lower DMSO concentration with pentastarch represents an improvement in the CB cryopreservation process and could have wider clinical application as an alternate freezing medium over 10% DMSO.

Circulating endothelial progenitor cells are not affected by acute systemic inflammation

Vascular injury causes acute systemic inflammation and mobilizes endothelial progenitor cells (EPCs) and endothelial cell (EC) colony-forming units (EC-CFUs). Whether such mobilization occurs as part of a nonspecific acute phase response or is a phenomenon specific to vascular injury remains unclear. We aimed to determine the effect of acute systemic inflammation on EPCs and EC-CFU mobilization in the absence of vascular injury. Salmonella typhus vaccination was used as a model of acute systemic inflammation. In a double-blind randomized crossover study, 12 healthy volunteers received S. typhus vaccination or placebo. Phenotypic EPC populations enumerated by flow cytometry [CD34(+)VEGF receptor (VEGF)R-2(+)CD133(+), CD14(+)VEGFR-2(+)Tie2(+), CD45(-)CD34(+), as a surrogate for late outgrowth EPCs, and CD34(+)CXCR-4(+)], EC-CFUs, and serum cytokine concentrations (high sensitivity C-reactive protein, IL-6, and stromal-derived factor-1) were quantified during the first 7 days. Vaccination increased circulating leukocyte (9.8 + or - 0.6 vs. 5.1 + or - 0.2 x 10(9) cells/l, P < 0.0001), serum IL-6 [0.95 (0-1.7) vs. 0 (0-0) ng/l, P = 0.016], and VEGF-A [60 (45-94) vs. 43 (21-64) pg/l, P = 0.006] concentrations at 6 h and serum high sensitivity C-reactive protein at 24 h [2.7 (1.4-3.6) vs. 0.4 (0.2-0.8) mg/l, P = 0.037]. Vaccination caused a 56.7 + or - 7.6% increase in CD14(+) cells at 6 h (P < 0.001) and a 22.4 + or - 6.9% increase in CD34(+) cells at 7 days (P = 0.04). EC-CFUs, putative vascular progenitors, and the serum stromal-derived factor-1 concentration were unaffected throughout the study period (P > 0.05 for all). In conclusion, acute systemic inflammation causes nonspecific mobilization of hematopoietic progenitor cells, although it does not selectively mobilize putative vascular progenitors. We suggest that systemic inflammation is not the primary stimulus for EPC mobilization after acute vascular injury.

Oxidized LDL-Activated Platelets Induce Vascular Inflammation

Platelets are involved in the initiation of atherosclerosis by adherence to inflamed endothelium. Monocytes bind to these platelets and transmigrate into the vessel wall, transforming into macrophages and foam cells. We have previously shown that lipid-laden platelets are phagocytosed by macrophages. In this study we investigated the functional consequences of oxidized low-density lipoprotein (oxLDL) uptake on platelet function and interaction with the endothelium. Human platelets were isolated from healthy donors and activated by adenosine diphosphate. Immunofluorescence microscopy and flow cytometry revealed that oxLDL is located intracellularly in vesicles. With mepacrine costaining and confocal microtomography, we were able to identify dense granules as the vesicles that contain oxLDL. OxLDL-laden platelets induced intercellular adhesion molecule 1 expression in endothelial cells more than exogenous native LDL, oxLDL, and oxLDL-negative platelets. Furthermore, oxLDL-laden platelets induced foam cell development from CD34(+) progenitor cells. On endothelial regeneration, oxLDL-laden platelets had the opposite effect: The number of CD34(+) progenitor cells (colony-forming units) able to transform into endothelial cells was significantly reduced in the presence of oxLDL-platelets, whereas native LDL had no effect. Our results demonstrate that activated platelets internalize oxLDL and that oxLDL-laden platelets activate endothelium, inhibit endothelial regeneration, and promote foam cell development. Platelet oxLDL contributes significantly to vascular inflammation and is able to promote atherosclerosis.

Correlations between progression of coronary artery disease and circulating endothelial progenitor cells

The pathophysiology of coronary artery disease (CAD) progression is not well understood. Endothelial progenitor cells (EPCs) may have an important role. In the present observational cohort study we assessed the number of circulating EPCs in 136 patients undergoing elective percutaneous coronary intervention and who had at least one major epicardial vessel with a nonsignificant stenosis [<50% diameter stenosis (DS)], and the relationship between plasma EPC levels and the 24-mo progression of the nonsignificant coronary artery lesion. The following cell populations were analyzed: CD34(+), CD133(+), CD34(+)/KDR(+), CD34(+)/VE cadherin(+), and endothelial cell colony-forming units (CFU-ECs). Progression was defined as a >15% DS increase of the objective vessel at follow-up. At 24 mo, 57 patients (42%) experienced significant progression. Independent predictors of disease progression were LDL cholesterol > 100 mg/dl (OR=1.03; 95% CI 1.01-1.04; P=0.001), low plasma levels of CFU-ECs (OR=3.99; 95% CI 1.54-10.37; P=0.005), and male sex (OR=3.42; 95% CI 1.15-10.22; P=0.027). Circulating levels of EPCs are significantly lower in patients with angiographic CAD progression.

MT1-MMP Is Required for Hematopoietic Maturation in the BM Niche.

Abstract 3634Poster Board III-570Specialized niches, in which hematopoietic stem cells (HSCs) reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. Activation of the fibrinolytic pathway via matrix metalloproteinase-9 (MMP-9) resulted in the release of kit ligand (KitL) in the BM niche. Membrane type 1-MMP (MT1-MMP) can activate e.g. MMP-9. To investigate the role of MT1-MMP in hematopoiesis, we used MT1-MMP deficient mice. MT1-MMP−/− mice examined 12 days after birth showed pancytopenia and reduced numbers of bone marrow mononuclear cells (BMMCs) and splenocytes. BM cytospins from MT1-MMP−/− mice showed mild perturbations in erythropoiesis and a more severe impairment of myelopoiesis. Although all lineages were present, the ratio of erythroid to myeloid precursors increased from 0.36 in wildtype to 0.60 in MT1-MMP−/− mice. Myeloid and erythroid cell differentiation was impaired in MT1-MMP−/− BMMCs. The numbers of colony forming unit cells (CFU-C) was reduced in MT1-MMP−/− BMMCs. In contrary, the number of immature hematopoietic cells (CFU-S8, KSL cells) was augmented in MT1-MMP−/− BMMCs. FACS analysis of BM cells showed a decrease in the percentage of mature B cells with an increase number of Pro-B and immature B cells in MT1-MMP−/− BMMCs relative to controls. MT1-MMP−/− BM cells showed lower expression of CXCL12 and KitL, typical niche growth factors important for myelopoiesis and lymphopoisis. Thus, MT1-MMP is required for normal hematopoietic differentiation of lymphoid and myeloid lineage cells, most likely due to growth factor defective niche cells. Disclosures:No relevant conflicts of interest to declare.

IL-27 Abrogates Receptor Activator of NF-κB Ligand-Mediated Osteoclastogenesis of Human Granulocyte-Macrophage Colony-Forming Unit Cells through STAT1-Dependent Inhibition of c-Fos

IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Ralpha (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-kappaB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-kappaB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.

Synergistic Effect of High Hydrostatic Pressure and Natural Antimicrobials on Inactivation Kinetics ofBacillus cereusin a Liquid Whole Egg and Skim Milk Mixed Beverage

An in-depth study was conducted in order to extend the storage life of a liquid whole egg-skim milk (LWE-SM) mixed beverage to enhance its safety and the safety of related beverages. Bacillus cereus vegetative cells (1 x 10(8) colony-forming units [CFU]/mL) were inoculated in LWE-SM beverages with or without natural antimicrobial supplements: flavonol rich-cocoa powder (cocoanOX 12%, CCX) (700 ppm), vanillin (700 ppm), anise (700 ppm), and cinnamon (700 ppm). B. cereus cells were maintained at 10 degrees C for 10 days in the different beverages to test the bacteriostatic or inhibitory effect of the aforementioned ingredients. Beverages were treated with high hydrostatic pressure (HHP) technology and stored at 10 degrees C for 15 days after treatment. All natural antimicrobials reduced the micro(max) values and increased the lag phase time of B. cereus, and Gompertz growth curves showed different inhibitory effects depending on the substance. The maximum inhibitory effect (1.330 log cycle reduction) was achieved in LWE-SM-cinnamon-supplemented beverage. The maximum inactivation achieved by HHP in LWE-SM beverage was a reduction of around 3.89 +/- 0.25 log cycles at 300 MPa for 12 minutes. When supplemented beverages were treated under the same conditions, enhanced inactivation levels were achieved. This increased inactivation can be attributed to a synergistic effect when the LWE-SM was supplemented with flavonol-rich cocoa powder, cinnamon, and vanillin. The maximum synergistic effect was observed in LWE-SM-CCX-supplemented beverage. During the refrigerated storage of B. cereus HHP-treated cells in beverages to which antimicrobials had been added, the inhibitory effect was dependent on the previously applied pressure level.

Correction of Murine Sickle Cell Disease Using γ-Globin Lentiviral Vectors to Mediate High-level Expression of Fetal Hemoglobin

Increased levels of red cell fetal hemogloblin, whether due to hereditary persistence of expression or from induction with hydroxyurea therapy, effectively ameliorate sickle cell disease (SCD). Therefore, we developed erythroid-specific, gamma-globin lentiviral vectors for hematopoietic stem cell (HSC)-targeted gene therapy with the goal of permanently increasing fetal hemoglobin (HbF) production in sickle red cells. We evaluated two different gamma-globin lentiviral vectors for therapeutic efficacy in the BERK sickle cell mouse model. The first vector, V5, contained the gamma-globin gene driven by 3.1 kb of beta-globin regulatory sequences and a 130-bp beta-globin promoter. The second vector, V5m3, was identical except that the gamma-globin 3'-untranslated region (3'-UTR) was replaced with the beta-globin 3'-UTR. Adult erythroid cells have beta-globin mRNA 3'-UTR-binding proteins that enhance beta-globin mRNA stability and we postulated this design might enhance gamma-globin expression. Stem cell gene transfer was efficient and nearly all red cells in transplanted mice expressed human gamma-globin. Both vectors demonstrated efficacy in disease correction, with the V5m3 vector producing a higher level of gamma-globin mRNA which was associated with high-level correction of anemia and secondary organ pathology. These data support the rationale for a gene therapy approach to SCD by permanently enhancing HbF using a gamma-globin lentiviral vector.

Near-optimal Conditions for the In Vitro Culture of Hemopoietic Progenitor Cells in Bone Marrow from the Rat

In vitro techniques for the culture of hemopoietic stem cells and committed hemopoietic progenitor cells in rat bone marrow have not been adequately described in the literature. In the present investigations, and using commercially available hemopoietic cytokines and growth factors, the conditions required to perform long-term bone marrow culture (LTBMC) using rat femoral bone marrow were studied, in conjunction with the colony-forming unit cell assay (CFU-C), to quantify the number of progenitor cells. CFU-C production by LTBMCs, set up using Iscove's modified Dulbecco's medium supplemented with fetal calf serum and horse serum, ceased after week 2 of culture. However, the duration of CFU-C production was significantly increased by supplementing LTBMCs with the cytokine recombinant mouse stem cell factor or recombinant rat stem cell factor.

Common endothelial progenitor cell assays identify discrete endothelial progenitor cell populations

Multiple measures of endothelial progenitor cells (EPCs) have been described, but there has been limited study of the comparability of these assays. We sought to determine the reproducibility of and correlation between alternative EPC assay methodologies. We simultaneously assessed EPC numbers in 140 patients undergoing cardiac catheterization using the 2 most commonly used culture techniques: endothelial cell outgrowth and colony-forming unit (CFU). In the final 77 patients, EPCs were also identified on the basis of cell surface marker expression (CD133, CD34, and vascular endothelial growth factor receptor-2 [VEGFR-2]) and aldehyde dehydrogenase (ALDH) activity. Endothelial progenitor cell enumeration based on fluorescence activated cell sorting was more precise than culture assays. There was limited correlation between EPC numbers determined using the 2 common culture-based assays; however, endothelial CFUs correlated with VEGFR-2 and CD34/VEGFR-2-expressing cells. Endothelial progenitor cells defined by expression of CD133, CD34, CD133/CD34, and ALDH activity correlated with each other, but not with VEGFR-2(+) cells. Endothelial progenitor cells can be broadly classified into 2 classes: VEGFR-2-expressing cells, which give rise to endothelial CFUs, and CD133/CD34 or ALDH(br) cells. These observations underscore the need for better assay standardization and a more precise definition of EPCs in cell therapy research.

Proteomics Identifies Thymidine Phosphorylase As a Key Regulator of the Angiogenic Potential of Colony-Forming Units and Endothelial Progenitor Cell Cultures

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin alphavbeta3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.

Osteoclast Function Is Essential in the Hematopoietic Stem Cell Niche

Hematopoietic stem cell (HSC) function and number are tightly regulated in a specific microenvironment - the HSC niche - constituted of several cell types. Amongst them, osteoblasts have a prominent role since their increment is associated with an expansion of HSC. However, we demonstrated that an overall increase in osteoblasts does not necessarily promote hematopoiesis (Lymperi et al, Blood 2008 111:1173-81). We hypothesised that osteoclast mediated bone resorption, a component of the bone remodelling process which regulates osteoblast formation, takes active part in the HSC niche. To address this question we inhibited osteoclast function with the bone catabolic agents bisphosphonates. In vivo administration of bisphosphonates to C57BL/6 mice altered the architecture of the trabecular bone as defined by micro-CT and histomorphometric analysis. Treated mice exhibited increased bone volume and trabecular number and reduced trabecular separation and trabecular pattern factor compared to the untreated controls. This was accompanied to a decrease in the proportion and absolute number of HSC in the bone marrow (BM) as assessed by both the immunophenotypic enumeration of Lin-Sca1+c-kit+Flk2-HSC and the long-term culture- initiating cell (LTC-IC) assay. The number of the colony forming unit-cells (CFU-C) in the BM of bisphosphonate treated mice was increased, while more Lin-Sca1+c-kit+ enriched cells were found in the S/M phase of the cell cycle. These results indicate that, in osteoclast-impaired mice a proportion of HSC enters the cell cycle changing the balance in favour of hematopoietic progenitors. The ability of BM cells from bisphosphonate treated mice to engraft and reconstitute the hematopoietic system was tested in a competitive transplantation assay, whereby BM cells from treated and untreated mice were transplanted into syngeneic irradiated recipients. The long-term engraftment of treated BM cells was inferior to the one of controls, indicating a decrease in HSC numbers. Furthermore, recipient mice treated with bisphosphonates prior to conditioning and BM transplantation experienced a delayed hematopoietic recovery as compared to untreated recipients. Since it has been shown that parathyroid hormone (PTH) administration increases the number of HSC by stimulating osteoblast numbers, mice receiving micro-pump delivered PTH were also injected with bisphosphonates. We observed that the administration of bisphosphonates abolished the ability of PTH to increase the primitive HSC pool size as measured by FACS analysis of the Lin-Sca1+c-kit+Flk2- BM cells and their capacity to engraft and reconstitute irradiated recipients. Our findings demonstrate that the osteoclast function is fundamental for the HSC niche, possibly by promoting the recruitment of osteoblasts with HSC supporting capacity.

Mobilization of Hematopoitic Stem/Progenitor Cells by a Cdc42 Activity-Specific Inhibitor

Hematopoietic stem cell transplantation has become a standard of care for the treatment of many hematological diseases. Transplantation of mobilized peripheral blood stem cells has replaced bone marrow (BM) transplantation as the preferred method for hematopoietic recovery. To date, G-CSF mobilized hematopoietic stem/progenitor cell (HSPC) harvest is the main FDA-approved preparative regiment for transplantation protocols, but this application has several limitations in utilities including diverse individual variability and potential side effects in several patient populations. Although AMD3100, a chemical CXCR4-blocker, has been found effective for HSPC mobilization, the development of additional HSPC mobilization agents that work through well defined molecular mechanisms remains in need. Previously our laboratory has shown in a conditional knockout mouse model that deficiency of the Rho GTPase Cdc42 in the BM causes impaired adhesion, homing, lodging and retention of HSPCs, leading to massive egress of HSPCs from BM to the peripheral blood without compromising their proliferative potential. From an array of small molecule inhibitors of PIP2-induced actin-polymerization discovered in a high throughput screening, we identified CASIN, a novel Cdc42 Activity-Specific Inhibitor, that is effective in suppressing Cdc42 activity in a dose-dependent manner in murine fibroblasts and low density bone marrow (LDBM) cells and human CD34+ umbilical cord blood (HCB) cells in vitro, and in murine LDBM cells in vivo. The inhibitory effect by CASIN appears to be specific to Cdc42 and is reversible. We subsequently tested the hypothesis that pharmacological targeting Cdc42 by CASIN may transiently mimic the Cdc42 knockout phenotype leading to HSPC mobilization. In the dose range of 5–10 uM, CASIN does not show detectable toxicity in wild type or Cdc42 knockout HSPCs in cell survival and colony-forming unit activity assays. CASIN treatment of 32D murine myeloid progenitor cells or freshly isolated progenitor cells results in a reversible inhibition of F-actin polymerization induced by SDF-1α and blockade of α5β1 integrin mediated adhesion to fibronectin fragment CH296. Its effects on actin organization and adhesion are associated with an inhibition of directional migration of the colony-forming cells toward SDF-1α. In contrast, CASIN does not show a detectable effect on the adhesion and migration activities of Cdc42 knockout HSPCs, suggesting that it works specifically through Cdc42 to affect cell actin structure and adhesion. Upon injection into mice (5mg/Kg, intraperitoneally), CASIN is effective in stimulating mobilization of progenitor activity into the peripheral blood (~ 6-fold increase compared to control at 40 hrs post injection). Subsequent serial transplantation experiments show that the PB harvested from CASIN treated mice could reconstitute various lineages of blood cells in primary, secondary, and tertiary recipients, indicating that long-term hematopoietic stem cells were mobilized from the BM of CASIN-treated donor mice. Consistent with the mobilization phenotype, FACS analysis shows that intravenous injection of CASIN can cause transient reduction of long-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34−) and short-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34+) from BM. Similar to the effects on murine HSPCs, CASIN is active on CD34+ HCB cells in transiently suppressing F-actin assembly, adhesion to fibronectin, and SDF-1α induced migration without detectable toxicity in vitro. Whether CASIN is effective in mobilizing HCB-engrafted NOD/SCID mice is currently under investigation. Our studies suggest that the novel concept of pharmacological targeting of Cdc42, that transiently and reversibly mimics the effect of Cdc42 knockout, may be developed into a mobilization regiment with a well defined molecular and cellular mechanism.

Bone marrow and tumor cell colony-forming units and human tumor xenograft efficacy of noncamptothecin and camptothecin topoisomerase I inhibitors

Topoisomerase I (TopoI), an established anticancer target, is an enzyme producing a single-strand DNA break during transcription. Several noncamptothecin TopoI inhibitors have been identified. One of these, ARC-111, was compared with two clinically used camptothecins, topotecan and irinotecan/SN-38. In mouse and human bone marrow colony formation [colony-forming units granulocyte-macrophage (CFU-GM)] assays, the IC(90) values were 519 and 331 nmol/L for topotecan and SN-38 mouse CFU-GM and were 19 and 26 nmol/L for human CFU-GM, giving mouse to human differentials of 28- and 13-fold. ARC-111 produced IC(90) values of 28 nmol/L in mouse and 6.2 nmol/L in human CFU-GM, thus only a 4.5-fold differential between species. Human bone marrow CFU-GM was more sensitive to topotecan than were several human cancer cell lines, but ARC-111 cytotoxicity was similar for human bone marrow CFU-GM and the seven human tumor cell lines tested. In HCT-116 xenografts, tumor growth delays (TGD) were 17 days for irinotecan and 20 days for ARC-111. In HT-29 xenografts, the TGD was 9 days for both irinotecan and ARC-111. Both ARC-111 and docetaxel had a TGD of 21 days in NCI-H460 xenografts, and both ARC-111 and gemcitabine had a TGD of 7 days in MiaPaCa2 xenograft. Current TopoI inhibitors have broad antitumor activity in human tumor xenografts that is not achieved in the clinic. This may be due to greater sensitivity of human bone marrow than mouse to the cytotoxicity of these agents. It may be possible to achieve similar levels of ARC-111 in patients as in mice allowing improved antitumor activity.

Hematopoietic progenitor cell colony growth differentiates chronic myelomonocytic leukemia from reactive monocytosis

In the absence of a cytogenetic abnormality or overt dysplasia, chronic myelomonocytic leukemia (CMML) may be difficult to be distinguished from reactive monocytosis. We have previously described a typical growth pattern in CMML patients, i.e., 'pseudonormal' colonies resembling granulocytic colonies but consisting entirely of monocytic cells when stained. To study the utility of the colony forming unit cell assay (CFU-C) as a diagnostic tool in patients with monocytosis, we analyzed a cohort of 48 consecutive patients referred to our institution with peripheral blood monocytosis. Thirty-six patients fulfilled the WHO criteria for CMML; 12 were diagnosed with reactive monocytosis. Of the patients with CMML, 28 showed pseudonormal growth with or without leukemic cluster growth, another four showed exclusively leukemic growth. None of the patients with reactive monocytosis showed either leukemic or pseudonormal growth. With a specificity of 100% and a sensitivity of 89%, the CFU-C assay has a unique potential to distinguish CMML from reactive monocytosis.

Assessing Identity, Phenotype, and Fate of Endothelial Progenitor Cells

From the paradigm shifting observations of Harvey, Malpighi, and van Leeuwenhoek, blood vessels have become recognized as distinct and dynamic tissue entities that merge with the heart to form a closed circulatory system.1 Vessel structures are comprised predominantly of a luminal layer of endothelial cells that is surrounded by some form of basement membrane, and mural cells (pericytes or vascular smooth muscle cells) that make up the vessel wall. In larger more complex vessel structures the vessel wall is composed of a complex interwoven matrix with nerve components. Understanding the cellular and molecular basis for the formation, remodeling, repair, and regeneration of the vasculature have been and continue to be popular areas for investigation. The endothelium has become a particularly scrutinized cell population with the recognition that these cells may play important roles in maintaining vascular homeostasis and in the pathogenesis of a variety of diseases.2 Although it has been known for several decades that some shed or extruded endothelial cells enter the circulation as apparent contaminants in the human blood stream,3 only more recent technologies have permitted the identification of not only senescent sloughed endothelial cells,4 but also endothelial progenitor cells (EPCs), which have been purported to represent a normal component of the formed elements of circulating blood5 and play roles in disease pathogenesis.6–9 Most citations refer to an article published in 1997 in which Asahara and colleagues isolated, characterized, and examined the in vivo function of putative EPCs from human peripheral blood as a major impetus for generating interest in the field.10 This seminal article presented some evidence to consider emergence of a new paradigm for the process of neovascularization in the form of postnatal vasculogenesis. Since publication of that article, interest in circulating endothelial cells, and particularly EPCs, has soared, …

Circulating Endothelial Progenitor Cells as a Pathogenetic Marker of Moyamoya Disease

Moyamoya disease (MMD) is an unusual form of chronic cerebrovascular occlusive disease that involves the formation of characteristically abnormal vessels. Recent studies have reported that colony-forming unit (CFU) and outgrowth cells represent a subpopulation of endothelial progenitor cells (EPCs). Here, we attempted to determine the significance of CFU number and outgrowth cell yield in MMD. Endothelial progenitor cells were isolated from the blood of 24 adult MMD patients and from 48 age- and risk factor-matched control subjects. After 7 days of culture, CFUs were determined, and yields of outgrowth cells were measured during 2 months of culture. The EPC function was also evaluated using matrigel plate assays. It was found that CFU numbers were significantly lower in MMD patients than in controls. Moreover, during long-term culture, outgrowth cells were isolated from only 10% of control subjects but from 33% of MMD patients, and CFU numbers and tube formation were found to be lower in advanced MMD cases than in those with early stage disease, whereas outgrowth cells were more frequently detected in those with early MMD and moyamoya vessels than in those with advanced disease. These characteristics of circulating EPCs reflect mixed conditions of vascular occlusion and abnormal vasculogenesis during the pathogenesis of MMD.

Leukemic Cluster Growth in Culture Is an Independent Risk Factor for Acute Myeloid Leukemia and Short Survival in Patients with Myelodysplastic Syndrome

In patients with myelodysplastic syndrome (MDS) precursor cell cultures (colony-forming unit cells, CFU-C) can provide an insight into the growth potential of malignant myeloid cells. In a retrospective single-center study of 73 untreated MDS patients we assessed whether CFU-C growth patterns were of prognostic value in addition to established criteria. Abnormalities were classified as qualitative (i.e. leukemic cluster growth) or quantitative (i.e. strongly reduced/absent growth). Thirty-nine patients (53%) showed leukemic growth, 26 patients (36%) had strongly reduced/absent colony growth, and 12 patients showed both. In a univariate analysis the presence of leukemic growth was associated with strongly reduced survival (at 10 years 4 vs. 34%, p = 0.004), and a high incidence of transformation to AML (76 vs. 32%, p = 0.01). Multivariate analysis identified leukemic growth as a strong and independent predictor of early death (relative risk 2.12, p = 0.03) and transformation to AML (relative risk 2.63, p = 0.04). Quantitative abnormalities had no significant impact on the disease course. CFU-C assays have a significant predictive value in addition to established prognostic factors in MDS. Leukemic growth identifies a subpopulation of MDS patients with poor prognosis.

Circulating Endothelial Progenitor Cells as a New Marker of Endothelial Dysfunction or Repair in Acute Stroke

Understanding on distinct subsets of endothelial progenitor cells may provide insights of endothelial dysfunction or repair in the acute ischemic event. Recent in vitro data have reported the colony-forming unit (CFU) and outgrowth cell population as a subset of endothelial progenitor cells. In this study, we undertook to validate the significance of CFU number and outgrowth cell yield in acute stroke. Mononuclear cells were isolated from the peripheral blood of 75 patients with acute stroke, 45 patients with chronic stroke, and 40 age-matched healthy volunteers. CFU numbers were counted after culturing them for 7 days, and outgrowth cell appearance was measured during the 2 months of culture. Endothelial progenitor cell function was also evaluated by matrigel plate assays. Independent parameters predicting CFU number and outgrowth cell yield were assessed using logistic regression analysis. The CFU numbers and tube formation abilities in matrigel assays were significantly reduced in patients with acute stroke compared with patients with chronic stroke or healthy control subjects. Moreover, patients with large artery atherosclerosis had much lower CFU numbers and functional activities than ones with cardioembolism. Outgrowth cells were isolated from 10% of healthy control subjects and 22% of patients with chronic stroke during the cultures, but from 71% of patients with stroke. Multivariate analysis identified glycosylated hemoglobin and National Institutes of Health Stroke Scale on admission as significant independent predictors of a low CFU number and a high isolation frequency of outgrowth cells, respectively. CFU number may thus represent an accumulated endothelial progenitor cell dysfunctional status, whereas outgrowth cell appearance may reflect the resilience of the systemic circulation to acute ischemic stress.