CD34-positive Stem Cells Research Articles

Arsenic exposure in utero exacerbates skin cancer response in adulthood with contemporaneous distortion of tumor stem cell dynamics.

Arsenic is a carcinogen with transplacental activity that can affect human skin stem cell population dynamics in vitro by blocking exit into differentiation pathways. Keratinocyte stem cells (KSC) are probably a key target in skin carcinogenesis. Thus, we tested the effects of fetal arsenic exposure in Tg.AC mice, a strain sensitive to skin carcinogenesis via activation of the v-Ha-ras transgene likely in KSCs. After fetal arsenic treatment, offspring received topical 12-O-tetradecanoyl phorbol-13-acetate (TPA) through adulthood. Arsenic alone had no effect, whereas TPA alone induced papillomas and squamous cell carcinomas (SCC). However, fetal arsenic treatment before TPA increased SCC multiplicity 3-fold more than TPA alone, and these SCCs were much more aggressive (invasive, etc.). Tumor v-Ha-ras levels were 3-fold higher with arsenic plus TPA than TPA alone, and v-Ha-ras was overexpressed early on in arsenic-treated fetal skin. CD34, considered a marker for both KSCs and skin cancer stem cells, and Rac1, a key gene stimulating KSC self-renewal, were greatly increased in tumors produced by arsenic plus TPA exposure versus TPA alone, and both were elevated in arsenic-treated fetal skin. Greatly increased numbers of CD34-positive probable cancer stem cells and marked overexpression of RAC1 protein occurred in tumors induced by arsenic plus TPA compared with TPA alone. Thus, fetal arsenic exposure, although by itself oncogenically inactive in skin, facilitated cancer response in association with distorted skin tumor stem cell signaling and population dynamics, implicating stem cells as a target of arsenic in the fetal basis of skin cancer in adulthood.

TNF-related apoptosis-inducing ligand (TRAIL) is expressed throughout myeloid development, resulting in a broad distribution among neutrophil granules

TRAIL induces apoptosis in a variety of tumor cells. Our laboratory found that human neutrophils contain an intracellular reservoir of prefabricated TRAIL that is released after stimulation with Mycobacterium bovis bacillus Calmette-Guérin. In this study, we examined the subcellular distribution of TRAIL in freshly isolated neutrophils. Neutrophil granules, secretory vesicles (SV), and plasma membrane vesicles were isolated by subcellular fractionation, followed by free-flow electrophoresis, and examined by ELISA and immunoblot. TRAIL was found in all membrane-bound fractions with the highest amounts in the fractions enriched in azurophilic granule (AG) and SV. Immunofluorescence confocal microscopy showed that TRAIL colocalized independently with myeloperoxidase (MPO), lactoferrin (LF), and albumin, respective markers of AG, specific granules, and SV. Furthermore, immunotransmission electron microscopy demonstrated that TRAIL colocalized intracellularly with MPO and albumin. We examined TRAIL expression in PLB-985 cells induced with dimethylformamide and in CD34-positive stem cells treated with G-CSF. Quantitative RT-PCR analysis showed that TRAIL was expressed in each stage of development, whereas MPO and LF were only expressed at distinct times during differentiation. Collectively, these findings suggest that TRAIL is expressed throughout neutrophil development, resulting in a broad distribution among different granule subtypes.

Positive selection for CD90 as a purging option in acute myeloid leukemia stem cell transplants

Several studies showed the benefit of purging of acute myeloid leukemia (AML) stem cell transplants. We reported previously that purging by positive selection of CD34+ and CD133+ cells resulted in a 3-4 log tumor cell reduction (TCR) in CD34- and/or CD133- AML, but has been shown to be potentially applicable in only about 50% of cases. Similar to CD34 and CD133, CD90 marks the hematopoietic CD34 positive stem cells capable of full hematopoietic recovery after myeloablative chemotherapy, and therefore, in the present study, we explored whether a similar purging approach is possible using CD90. CD90 expression was established by flowcytometry in diagnosis AML on the clonogenic AML CD34+ blast population by flow cytometry. Positivity was defined as >3% CD90 (CD34+) expression on blasts. For the calculation of the efficacy of TCR by positive selection, AML blasts were recognized by either prelabeling diagnosis blasts with CD45-FITC in spiking model experiments or using expression of leukemia associated marker combinations both in spiking experiments and in real transplants. In 119 patients with AML and myelodysplastic syndrome, we found coexpression of CD34 and CD90 (>3%) in 42 cases (35%). In AML patients 60 years or younger, representing the patients who are eligible for transplantation, only 23% (16/69) of the patients showed CD90 expression. Positive selection for CD90 in transplants containing CD90 negative AML resulted in a 2.8-4 log TCR in the models used. Purging by positive selection using CD90 can potentially be applied effectively in the majority of AML patients 60 years or younger.

Interferon-α2b-Induced Thrombocytopenia Is Caused by Inhibition of Cytoplasmic Maturation and Platelet Production, but Not Proliferation and Endomitosis in Human Megakaryocytes.

Human interferon (IFN)-α is a standard treatment for the patients with chronic hepatitis C. It is well known that chronic hepatitis C can transform into liver cirrhosis and hepatocellular carcinoma, and the risk of progression to liver cirrhosis is around 40% in 5 years. Thus, it is important for chronic hepatitis C patients to receive IFN-α treatment to prevent its malignant transformation. Thrombocytopenia is one of the major adverse effects of IFN-α and often leads to a dose reduction or discontinuation of IFN-α therapy. However, there is little information how IFN-α inhibits human megakaryopoiesis. In this study, we demonstrated that IFN-α does not inhibit colony formation of megakaryocytes (CFU-MK) from human CD34-positive hematopoietic stem cells. IFN-α also does not inhibit endomitosis (DNA duplication without cytokinesis), but inhibits cytoplasmic maturation of megakaryocytes and platelet production in vitro. The inhibitory effects of IFN-α on the development of demarcation membrane in human megakaryocytes were visualized by staining primary megakaryocytes with di-8-ANEPPS dye under a confocal laser microscope. Loss of platelet production by IFN-α was analyzed by measuring proplatelet formation (PPF) and counting the human platelets produced in the supernatant of human primary megakaryocytes by flow cytometry. The expression of transcription factors regulating late stage megakaryopoiesis such as GATA-1, p45 NF-E2 and MafG was suppressed by IFN-α. To confirm these in vitro observations in vivo system, we transplanted human CD34-positive hematopoietic cells into the immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice (hu-NOG) after 2.4 Gy irradiation. Recombinant human(rh) IFN-α significantly reduced the number of human platelets but did not decrease the number of human megakaryocytes in hu-NOG mice. rhIFN-α did not change murine platelet counts in hu-NOG mice, as IFN-α has species specificity. The DNA ploidy of human megakaryocytes in the bone marrow of hu-NOG mice was not altered after treatement with rhIFN-α. We also confirmed the life time of human platelets was not shortend by rhIFN-α in hu-NOG mice, indicating IFN-α does not promote the clearance of human platelets. We have also confirmed that a novel thrombopoietin mimetics, NIP-004, prevented rhIFN-α-induced thrombotytopenia and increased the number of polyploid human megakaryocytes in hu-NOG mice, suggesting NIP-004 might be useful for the patients with hepatitis C to avoid IFN-α-induced thrombocytopenia. In conclusion, these results clarified that IFN-α induces thrombocytopenia by inhibiting the cytoplasmic maturation of megakaryocytes but not proliferation and endomitosis in human megakaryocytes.

Capture and Enrichment of CD34-Positive Haematopoietic Stem and Progenitor Cells from Blood Circulation Using P-Selectin in an Implantable Device.

Clinical infusion of haematopoietic stem and progenitor cells (HSPCs) is vital for restoration of haematopoietic function in many cancer patients. Previously we have demonstrated an ability to mimic physiologic cell trafficking in order to capture CD34-positive (CD34+) HSPCs using a monolayer of P-selectin in flow chambers. The aim of the current study was to determine if HSPCs could be captured directly from circulating blood in vivo. Vascular shunt prototypes, coated internally with P-selectin, were inserted into the femoral artery of rats. After one-hour blood perfusion, successful capture of mononuclear cells was achieved and analysis of these cells revealed a significant enrichment for CD34+ HSPCs. Blood flow through the cell capture device resulted in a wall shear stress of 4–6 dynes/cm2. Purity of captured CD34+ cells showed 7-fold enrichment (average purity = 28 ± 4%) over levels found in the control mobilized peripheral blood mononuclear cells (average purity = 3 ± 0.5%). Robust cell capture and HSPC enrichment were also observed in devices that were implanted in a closed-loop arterio-venous shunt conformation for two hours. Adherent cells were viable and were able to differentiate into burst forming units in culture. This study demonstrates an ability to mimic the physiologic arrest of HSPCs from blood in an implantable device and may represent a practical alternative for adult stem cell capture and enrichment.

Long-Term Survival after Reduced Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation in AML-Patients Is Consistently Sustained and Influenced by Donor Type Selection.

A total of 46 patients (24 female, 22 male) with acute myeloid leukemia (AML) received RIC allogeneic blood stem cell transplants from either fully HLA-compatible family donors (n=14) or unrelated HLA-compatible donors (n=32) between 3/1999 and 3/2007. The unselected consecutive single centre study patients (median age: 52 years, range: 22–64) were at high risk for treatment-related complications (older age, previous infectious complications, impaired organ function). 67% (n=31) of the cohort were not in first remisson but in an advanced status of AML. All patients received 30 mg·m−2 Fludarabine i.v. day −10 to −5, Busulfan 4mg·kg−1 orally day −5 to −4, and 10 mg·kg−1 i.v. Antithymocyteglobulin (ATG-Fresenius®) day −4 to −1 as RIC prior to allogeneic stem cell transplantation. A median of unselected 6.8 x 106 CD-34 positive stem cells (range: 1.9–14.3) were given. GvHD-prophylaxis consisted of Cyclosporin 3mg·kg−1 continuous infusion in case of 10/10 HLA compatibility donor situation (n=34), with the addition of mycophenolate mofetil 2gr. i.v. primarily starting from day +10 in one antigen mismatched HLA-unrelated donor situation (n=12). Nine patients (19.6%) died of therapy related mortality, due to GvHD (n=3), infections (n=4), or both (n=2). Relapses occurred in 18 patients (39%), in five patients even more than one year after transplantation. The median observation time of the study group was 19 months (range: 3–101), the product-limit estimates (Kaplan-Meier) of overall survival (OS) at 36 months were 0.48 (±0.08), of event-free survival (EFS) 0.44 (±0.08), respectively. [Display omitted] Disease-status (remisson vs. advanced) failed to have a significant impact in OS or EFS, whereas donor status (related vs. unrelated) resulted in OS at 24 months 0.76 (±0.12) for related donors and 0.4 (±0.09) for unrelated donors (p=0.017, log-rank test). RIC with Fludarabine/Busulfan/ATG prior to allogeneic stem cell transplantation results in a consistently sustained long-term outcome for this otherwise adverse patient subgroup, favouring family-matched donor selection.

Identifikation und Charakterisierung von hoch-metastatischen und therapie-resistenten Tumorstammzellen beim Pankreaskarzinom

Introduction: Despite our advancing knowledge of tumor cell biology, the treatment efficacy in most cancers has not significantly improved over the past decades. Accumulating evidence suggests that cancer may actually be a disease of stem cells. It has been shown that a small fraction of cancer cells bear stem cells properties and one possible explanation would be that these cancer stem cells (CSC) are derived from normal tissue stem cells. So far, however, in solid tumors these CSC have only been described for brain and breast cancer and their biological properties with respect to tumor progression remain enigmatic to date. Therefore, the purpose of the present study was to identify CSC in pancreatic cancer and define their role in tumor metastasis. Material and Methods: Putative cancer stem cells were identified by flow cytometry using the stem cell marker prominin-1 (CD133) and isolated by means of magnetic microbeads cell sorting. We analyzed pancreatic tissue as well as pancreatic tumors from patients with pancreatic cancer. Moreover, we evaluated non-metastatic human FG pancreatic cancer cells and highly-metastatic human L3.6pl pancreatic cancer cells with respect to their CSC content in vitro and in vivo. The functional activity of the cells was assessed in vitro using a modified Boyden chamber assay (membrane coated with Matrigel™ and in vivo using a murine model of orthotopic pancreatic cancer (female 8 week old NMRI nu/nu mice). Results: Human pancreatic cancer tissue contained a variable, but distinct CD133 positive CSC population ranging from 1 to 3% of the total cell count (n=3). SELDI-TOF mass spectrometry analysis indicated a marked overlap in the protein expression pattern between pancreatic stem cells and pancreatic cancer stem cells supporting the hypothesis that CSC are indeed derived from tissue resident stem cells. Cultured non-metastatic human FG pancreatic cancer cells contained only very few CD133 positive cells (0.02%) whereas highly-metastatic human L3.6pl pancreatic cancer cells were characterized by a distinct CD133 positive stem cells population ranging from 1 to 2% (n=5 each). The migratory capacity of isolated CSC from L3.6pl tumors was significantly higher (+136%) as compared to CD133 negative tumor cells (P<0.01) and primarily mediated by the CXCR4 receptor. A significantly lower migratory activity was observed for cancer cells isolated from FG tumors with no difference between CD133 positive and CD133 negative cells. After orthotopic implantation in NMRI nu/nu mice, the developed tumors derived from FG cells contained a very small population of CD133 positive cells (0.2 to 0.4%; n=8) whereas tumors derived from L3.6pl cells include a robust CD133 pos CSC population representing about 2% of the tumor cell population (n=10). Intriguingly, as few as 1,000 CD133 positive CSC isolated from L3.6pl tumors were capable of inducing new tumors (5/8 animals) whereas no tumor developed in mice receiving 103 CD133 negative tumors (0/8 animals). In 4 out of 5 animals with developing tumors after injection of CD133 positive CSC, metastatic lesions were observed. Although the anti-metabolite gemcitabine was capable of inducing apoptosis of CD133 negative tumor cells, it completely failed to induced apoptosis or cell death in CD133 positive CSC. Long-term in vitro treatment produced tumor cells that were markedly enriched for tumor stem cells. In vivo, gemcitabine significantly reduced tumor burden but also did not affect cancer stem cells resulting in a 7.6-fold increase in the relative stem cell content as compared to untreated animals. Conclusions: Here we demonstrate that human pancreatic cancers contain a low, but persistent percentage of CD133 positive cancer stem cells with highly invasive characteristics. Consistently, the highly-invasive human L3.6pl pancreatic cancer cells also contained a distinct cancer stem cell population whereas in non-metastatic FG pancreatic cancer cells this population was hardly detectable. The isolated CSC demonstrated a strong tumor repopulating capacity as well as a marked invasive activity which may explain at least in part the different metastatic activity of these two cell types. Most importantly, cancer stem cells are resistant to standard treatment. Thus, cancer stem cells represent a challenging novel target for the development of advanced molecular and pharmaceutical therapeutics.

Immunohistochemical investigation of CD34 antigen in male breast carcinoma

A total of 30 archival cases of male breast carcinoma were studied immunohistochemically for the expression of CD34 antigen. An obvious CD34 staining was found in three cases. By adding the original CD34-positive case, recently published as unique CD34-stained male breast carcinoma, the number of positive cases comes to four. This case was classified as an invasive papillary carcinoma of solid conformation, whereas the three other cases were invasive ductal carcinomas, not otherwise specified. The aim of this study is to establish whether the CD34 positivity, observed in the case of papillary subtype, was a case-specific finding. CD34 expression in the male breast carcinoma, according to our findings, seems to be neither a feature presented exclusively by a singular case nor a specific immunophenotype characterising a special type. The presence or preservation of CD34 antigen in four totally male breast carcinomas may be considered as a novel finding that supports a relationship between these tumours and the progenitor CD34-positive stem cells, committed to the organogenesis of mammary gland. In this context we hypothesise an origin of male breast carcinoma from the stem cells expressing or not the CD34 antigen according to their stage of differentiation.

Foamy Virus Vectors: An Awaited Alternative to Gammaretro- and Lentiviral Vectors

The first vectors derived from foamy viruses were established over ten years ago. Until now only used and further developed by a handful of investigators these vectors have been shown to be promising tools for the gene transfer into haematopoietic stem cells. Several inherent features of foamy virus-derived vectors, such as the high efficiency in targeting CD34-positive stem cells, a favourable integration profile, and the apathogenic nature of the parental virus, indicate that they are superior to gammaretroviral and lentiviral vectors. The effectiveness in different preclinical animal models suggests the exploration of foamy virus vectors in human trials.

Quiescent Subpopulations of Human CD34-Positive Hematopoietic Stem Cells Are Preferred Targets for Stable Recombinant Adeno-Associated Virus Type 2 Transduction

We have previously demonstrated recombinant adeno-associated viral (rAAV) transduction of human CD34(+) hematopoietic stem cells (HSCs) capable of serial engraftment in vivo. Here we evaluated the capacity of rAAV2 to mediate gene transfer into nondividing, quiescent, primitive CD34(+) cells subdivided on the basis of metabolic, mitotic, and phenotypic properties. Results revealed that CD34(+)CD38() marrow cells are the most quiescent, exist primarily in G(0) at isolation and are the only population to remain nondividing during the entire exposure to free rAAV. Despite significant differences in the extended clonogenic capacities of CD34(+) subsets in stromal cultures, the frequency of rAAV marking of colonies derived from primitive progenitors was similar in all three populations, suggesting that both primitive and more differentiated progenitors were initially transduced at equal levels. After transduction, episomal and integrated rAAV genomes were detected in all CD34(+) subsets. However, the more quiescent cells displayed higher levels of integrated rAAV than did rapidly dividing cells. Importantly, stable long-term integration was observed only in the most primitive, quiescent CD34(+)CD38(-) subset, indicating that this HSC compartment comprises the preferred substrate for stable rAAV2 transduction. Previously described rate limitations to transgene expression were observed in transduced CD34(+) cells and could be overcome by tyrphostin pretreatment, which resulted in augmented second-strand synthesis. These results represent the first demonstration of rAAV-mediated gene transfer to primitive, quiescent human CD34(+)CD38(-) stem cells and reveal that nondividing CD34(+)CD38(-) HSCs are the optimal CD34(+) targets for rAAV transduction.

Effect of Fibroblast Growth Factor 2 on Stromal Cell-Derived Factor 1 Production by Bone Marrow Stromal Cells and Hematopoiesis

Reduction of intramedullary hematopoiesis and the development of myelofibrosis and splenic hematopoiesis are frequent complications of clonal myeloid disorders that cause severe morbidity and death and present a therapeutic challenge. However, the pathogenesis of these complications is still unknown. We evaluated the effect of fibroblast growth factor 2 (FGF-2), the level of which is elevated in patients with clonal myeloid disorders, on bone marrow stromal cell expression of stromal cell-derived factor 1 (SDF-1), a chemokine that is essential for normal hematopoiesis. Reverse transcription-polymerase chain reaction analysis, immunoblot analysis, and enzyme-linked immunosorbent assays were used to examine effects of human recombinant FGF-2 exposure on SDF-1 expression in mouse stromal MS-5 and S-17 cells. Cocultures of human CD34-positive peripheral blood stem cells or mouse pre-B DW34 cells with mouse stromal cells were used to characterize the functional relevance of the effects of FGF-2 on SDF-1 expression. The in vivo hematologic effects of FGF-2 were determined by systemic administration to mice (n = 10). All statistical tests were two-sided. FGF-2 reduced constitutive SDF-1 mRNA expression and secretion in stromal cells (SDF-1 levels in supernatants: MS-5 cells cultured for 3 days in medium only versus in medium with FGF-2, 95.4 ng/mL versus 22.2 ng/mL, difference = 73.2 ng/mL, 95% confidence interval [CI] = 60.52 to 85.87 ng/mL; P = .002, two-sided Student's t test; S-17 cultured in medium only versus in medium with FGF-2, 203.53 ng/mL versus 32.36 ng/mL, difference = 171.17 ng/mL, 95% CI = 161.8 to 180.6 ng/mL; P<.001). These effects of FGF-2 were reversible. FGF-2 compromised stromal cell support of the growth and survival of pre-B DW34 and myeloid lineage cells, and these effects were reversed in part by exogenous recombinant SDF-1alpha (rSDF-1alpha) (DW34 pre-B cells recovery on S-17 stromal cells, expressed as a percentage of DW34 cells recovered from medium only: with FGF-2 versus without FGF-2, 27.6% versus 100%, difference = 72.4%, 95% CI = 45.34% to 99.51%, P = .008; with FGF-2 plus rSDF1 versus with FGF-2 only, 60.3% versus 27.6%, difference = 32.7%, 95% CI = 9.35% to 56.08%, P = .034; fold increase in number of myeloid lineage cells after culture on S-17 stromal cells: with FGF-2 versus without FGF-2, 0.25-fold versus 3.8-fold, difference = 3.55-fold, 95% CI = 2.66- to 4.44-fold, P<.001; recovery of myeloid cells on S-17 stromal cells, expressed as a percentage of myeloid cells recovered from medium only: FGF-2 plus rSDF-1alpha versus FGF-2 only, 76.5% versus 32.4%, difference = 44.1%, 95% CI = 32.58% to 55.68%, P<.001). Administration of FGF-2 to mice reversibly reduced bone marrow levels of SDF-1 and cellularity and induced immature myeloid cell mobilization, extramedullary hematopoiesis, and splenomegaly. Systemic administration of FGF-2 in mice disrupts normal bone marrow hematopoiesis in part through reduced expression of SDF-1. Thus, endogenous FGF-2 may represent a potential therapeutic target in clonal myeloid disorders characterized by bone marrow failure.

118: Co-transplanation of haploidentical mesenchymal stem cells to overcome graft dysfunction associated with parental haploidentical CD34 postive selected peripheral stem cell grafts

Transplantation with haploidentical peripheral blood stem cells from a parent are an acceptable form of treatment for children who require stem cell transplantation but lack a conventional donor. However, the risk of graft dysfunction increases due to the intense T cell depletion of CD34 positive stem cell selection. Mesenchymal stem cells (MSCs) isolated from bone marrow can improve hematological recovery but their role in overcoming graft dysfunction post transplant is unknown.

Combination High-Dose Cyclophosphamide and Bortezomib Is Safe and Effective for Stem Cell Harvesting in Chemotherapy Refractory Multiple Myeloma.

Introduction: High-dose melphalan and autologous stem cell transplantation is the accepted therapy for most patients with multiple myeloma (MM) following steroid-based induction therapy. In a significant proportion of patients, however, the disease is refractory to standard induction. The use of dose-intense combination chemotherapy, such as D-PACE (dexamethasone, doxorubicin, cyclophosphamide, and cisplatin), may affect the ability to harvest an adequate number of hematopoeitic stem cells prior to transplantation. In addition, in those patients not achieving adequate cytoreduction despite combination chemotherapy, there is a theoretical risk of stem cell product contamination by malignant plasma cells. Bortezomib is a therapeutic agent with a novel mechanism of action, which in preliminary studies appears to be synergistic to alkylating agents and does not appear to affect stem cell yield. We piloted the addition of bortezomib to high-dose cyclophosphamide during stem cell harvesting in a series of patients failing to achieve an adequate response to D-PACE salvage.Patients and Methods: Between 2002 and 2006, fifteen MM patients refractory to standard dexamethasone-based induction therapy received ≥ 2 cycles of D-PACE prior to proceeding to autologous stem cell harvest and transplantation. 7/15 patients achieved adequate cytoreduction and proceeded to high-dose cyclophosphamide (3 g/m2) and filgrastim plus ancestim stimulation for stem cell mobilization. However, 8 patients in this cohort did not achieve adequate disease cytoreduction following D-PACE. Therefore, bortezomib was added to the mobilization regimen on days 1, 4, 8, and 11, in addition to high-dose cyclophosphamide given on day 11. Identical growth factor stimulation was provided. Response assessment included days to stem cell harvest, number of CD34 cells harvested, plasma cells in the product, disease response, and hematologic parameters.Results: Pre-treatment toxicities from D-PACE were similar in both groups. The addition of bortezomib to cyclophosphamide during stem cell mobilization did not lead to increased symptomatic toxicity. Grade 3/4 thrombocytopenia occurred in 5/8 patients receiving combination bortezomib/cyclophosphamide. No episodes of significant bleeding, peripheral neuropathy, or skin rash were noted. The average CD34-positive stem cell harvest in both groups was >5.0 × 106/kg. Time to stem cell harvesting was not significantly different between the groups. Flow cytometric examination of the harvested product from the bortezomib/cyclophosphamide group consistently demonstrated <2% cells bearing plasma cell markers. One patient in each group failed to mobilize sufficient stem cells. Bone marrow plasmacyte counts following combination therapy and harvesting decreased in all assessed patients. Time to engraftment was similar in both groups. Post-transplant disease control and survival remains to be assessed, as some patients in the combination group have only recently undergone transplantation.Conclusion: The addition of bortezomib to high-dose cyclophosphamide during stem cell mobilization does not increase toxicity or decrease stem cell harvest yield or quality, and appears to achieve adequate disease reduction in patients otherwise refractory to combination chemotherapy. This may result in improved relapse-free survival in patients with refractory MM.

Circulating endothelial progenitor cells are inversely correlated with the median oxygen tension in the tumor tissue of patients with cervical cancer

Tumor hypoxia leads to adaptive responses in cancer cells, including an induction of vasculogenesis initiated by circulating endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs). The aim of the present study was to correlate the number of EPCs and CECs with the oxygenation of cervical cancer. Blood concentrations of EPCs were detected by FACS analysis with antibodies for CD34 and vascular endothelial growth factor receptor 2 (VEGFR2). CECs were evaluated by double staining for 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Di-LDL) and lectin in a cell culture assay. Ten patients with cervical cancer were compared with ten healthy volunteers. Intratumoral oxygen tension was assessed polarographically with the computerized Eppendorf histography system. Analysis of CEC numbers revealed no difference between patients and controls. However, patients had lower concentrations of CD34-positive hematopoietic stem cells (HSCs) but a significantly higher fraction of EPCs related to the number of HSCs (1.09% versus 0.53%). This fraction was significantly inversely correlated to the median oxygen tension (r = -0.74, p = 0.015). Our study shows for the first time a significant inverse correlation between the fraction of EPCs and intratumoral oxygen tension. We conclude that the fraction of EPCs should be further evaluated as a useful and convenient marker in the prediction of tumor tissue oxygenation.

Potentiation of the Antitumor Effect of Merocyanine 540‐mediated Photodynamic Therapy by Amifostine and Amphotericin B

Leukemia and lymphoma cells are much more sensitive to Merocyanine 540 (MC540)-mediated photodynamic therapy (PDT) than normal pluripotent hematopoietic stem cells and normal colony forming unit-granulocyte/macrophage progenitors (CFU-GM). By contrast, most solid tumor cells are only moderately sensitive to MC540-PDT. The limited activity against solid tumor cells has detracted from MC540's appeal as a broad-spectrum purging agent. We report here that noncytotoxic concentrations of amifostine (Ethyol, Ethiofos, WR-2721) and amphotericin B used either alone or in combination potentiate the MC540-sensitized photoinactivation of leukemia cells, wild-type small cell lung cancer cells and cisplatin-resistant small cell lung cancer cells. Amphotericin B also enhances the MC540-sensitized photoinactivation of normal CFU-GM, whereas amifostine protects CFU-GM against the cytotoxic action of MC540-PDT. The yield of CD34-positive normal hematopoietic stem and progenitor cells is only minimally diminished by pretreatment with amifostine, amphotericin B or combinations of amifostine plus amphotericin B. Purging protocols that combine MC540-PDT with amifostine or with amifostine plus amphotericin B could offer a simple and effective approach to the purging of autologous stem cell grafts that are contaminated with solid tumor cells or the purging of stem cell grafts from heavily pretreated leukemia patients that contain reduced numbers of normal stem and progenitor cells and, therefore, can ill afford additional losses caused by purging.

Prevention of Left Ventricular Remodeling With Granulocyte Colony-Stimulating Factor After Acute Myocardial Infarction

Experimental and clinical evidence has recently shown that pluripotent stem cells can be mobilized by granulocyte colony-stimulating factor (G-CSF) and may enhance myocardial regeneration early after primary percutaneous coronary intervention (PCI) management of acute myocardial infarction. Sustained or long-term effects of mobilized CD34-positive mononuclear stem cells, however, are unknown. Thirty consecutive patients with ST-elevation myocardial infarction undergoing primary PCI with stenting and abciximab were selected for the study 85+/-30 minutes after PCI; 15 patients were randomly assigned to receive subcutaneous G-CSF at 10 microg/kg body weight for 6 days in addition to standard care including aspirin, clopidogrel, an angiotensin-converting enzyme inhibitor, beta-blocking agents, and statins. In patients with comparable demographics and clinical and infarct-related characteristics, G-CSF stimulation led to sustained mobilization of CD34 positive mononuclear cells (MNC(CD34+)), with a 20-fold increase (from 3+/-2 at baseline to 66+/-54 MNC(CD34+)/microL on day 6; P<0.001); there was no evidence of leukocytoclastic effects, accelerated restenosis rate, or any late adverse events. Within 4 months, G-CSF-induced MNC(CD34+) mobilization led to enhanced resting wall thickening in the infarct zone of 1.16+/-0.29 mm (P<0.05 versus control), which was sustained at 1.20+/-0.28 after 12 months (P<0.001 versus control). Similarly, left ventricular ejection fraction improved from 48+/-4% at baseline to 54+/-8% at 4 months (P<0.005 versus control) and 56+/-9% at 12 months (P<0.003 versus control and paralleled by sustained improvement of wall-motion score index from 1.70+/-0.22 to 1.42+/-0.26 and 1.33+/-0.21 at 4 and 12 months, respectively), after G-CSF (P<0.05 versus baseline and P<0.03 versus controls). Accordingly, left ventricular end-diastolic diameter showed no remodeling and stable left ventricular dimensions after G-CSF stimulation, whereas left ventricular end-diastolic diameter in controls revealed enlargement from 55+/-4 mm at baseline to 58+/-4 mm (P<0.05 versus baseline) at 12 months after infarction and no improvement in diastolic function. Mobilization of MNC(CD34+) by G-CSF after primary PCI may offer a pragmatic strategy for improvement in ventricular function and prevention of left ventricular remodeling 1 year after acute myocardial infarction.

In vitro hematopoietic differentiation of human embryonic stem cells induced by co‐culture with human bone marrow stromal cells and low dose cytokines

Human embryonic stem (hES) cells randomly differentiate into multiple cell types during embryoid body (EB) development and limited studies have focused on directed hematopoietic differentiation. Here, we report that the treatment of hES cells during EBs development with a combination of low dose hematopoietic cytokines, including stem cell factor (SCF), Flt-3 ligand, vascular endothelial growth factor (VEGF) and human bone marrow stromal cells (hBMSCs), generated cell clusters that contained 8.81% KDR-positive hemangioblasts, 9.94% CD34-positive hematopoietic stem cells and 25.7% CD45-positive mature hematopoietic cells, and expressed hematopoietic genes such as KDR, stem cell leukemia (scl) and runt-related transcription factor 1 (Runx1). We provide the first evidence for the role of the cytokine-hBMSCs combination in promoting hematopoietic differentiation of hES cells, and thus provide the potential for generation of hematopoietic cells, as well as for understanding early developmental events that govern the initiation of hematopoiesis in humans.

Severe early acute humoral rejection resulting in allograft loss in a renal transplant recipient with Campath-1H induction therapy

One of the major goals in organ transplantation is to reduce immunosuppression by reprogramming the immune system so as to encourage tolerance. This process has been shown in animal models to be facilitated by lymphocyte depletion at the time of the transplant, and the degree of lymphocyte depletion substantially impacts the efficacy of such treatment [1,2]. Campath-1H is a humanized CD52-specific depleting complement-fixing cytotoxic IgG1 monoclonal antibody. CD52 antigen is located on the surface of T and B lymphocytes, natural killer (NK) cells, and less densely on monocytes [3]. Campath-1H profoundly depletes T lymphocytes from the peripheral blood for several months as well as having less marked effects on B lymphocytes, NK cells, monocytes and CD34-positive immature stem cells [3]. Campath-1H was initially used to treat acute renal allograft rejection [4] and later used successfully together with low dose cyclosporin (CsA) as induction therapy in renal transplantation [3]. Several other centres have also now reported their experiences with the use of Campath-1H together with various combinations of immunosuppressive agents in renal transplantation [5–9]. In some of the series there was a higher than usual incidence of acute humoral or antibody-mediated rejection (AHR) among the acute rejection (AR) episodes [6,8]. We report a case of early severe AHR resulting in renal allograft loss in a patient treated with Campath-1H induction therapy. Case

Elemental Selenium Generated by the Photobleaching of Seleno-Merocyanine Photosensitizers Forms Conjugates with Serum Macro-Molecules That are Toxic to Tumor Cells

Elemental selenium generated by the photobleaching of selenomerocyanine dyes forms conjugates with serum albumin and serum lipoproteins that are toxic to leukemia and selected solid tumor cells but well tolerated by normal CD34-positive hematopoietic stem and progenitor cells. Serum albumin and lipoproteins act as Trojan horses that deliver the cytotoxic entity (elemental selenium) to tumor cells as part of a physiological process. They exploit the fact that many tumors have an increased demand for albumin and/or low-density lipoprotein. Se(0)-protein conjugates are more toxic than selenium dioxide, sodium selenite, selenomethionine, or selenocystine. They are only minimally affected by a drug resistance mechanism, and they potentiate the cytotoxic effect of ionizing radiation and several standard chemotherapeutic agents. The cytotoxic mechanism of Se(0)-protein conjugates is not yet fully understood. Currently available data are consistent with the notion that Se(0)-protein conjugates act as air oxidation catalysts that cause a rapid depletion of intracellular glutathione and induce apoptosis. Drugs modeled after our Se(0)-protein conjugates may prove useful for the local and/or systemic therapy of cancer.

O-28 DNA microarray analysis ofmyelodysplastic syndromes with purified CD133-positive stem cell fractions

O-28 DNA microarray analysis ofmyelodysplastic syndromes with purified CD133-positive stem cell fractions