Biomedical subjects

Axel R Zander

Publications and source records attributed to Axel R Zander.

35 records · Page 2Linked to original sources

PubMed2004

Autologous serum for isolation and expansion of human mesenchymal stem cells for clinical use.

OBJECTIVE: Mesenchymal stem cells (MSC) are promising candidates for cell-based therapies. One major obstacle for their clinical use is the biosafety of fetal calf serum (FCS), which is a crucial part of all media currently used for the culture of MSC. METHODS: Nine donors each contributed 5 mL of bone marrow aspirate. Isolation of MSC was conducted according to Caplan et al., although for expansion we used low-density seeding with 20 MSC/cm2. Four different media A, B, C, and D were tested, containing 1%, 3%, or 10% autologous serum (AS), or 10% selected FCS, respectively. MSC were cultured on 24-well plates until passage 2 and counted under the microscope at regular intervals. Osteogenic and adipogenic differentiation were induced in vitro by using a modified standard cocktail and were evaluated semi-quantitatively through a microscope. RESULTS: Isolation of MSC after 3 days appeared best in media C with almost always C>D congruent with B>A. Proliferation was exponential with generally C>D>B>A. Morphologically, MSC isolated and expanded in medium C were indistinguishable from those in medium D. Phenotypic markers of MSC grown in medium C were: CD34-, CD45-, CD90+, CD105+, MHC class I+, MHC class II-, similar to MSC isolated and grown in medium D. Moreover, MSC grown in medium C showed more osteogenic potential than those from medium D in all cases: C+++, D++, B+, A 0. Cells retained their immaturity as shown by adipogenic differentiation and it always was: D+++, C++, B+, A 0. CONCLUSIONS: Growth of MSC in a FCS-free medium is feasible without addition of growth factors. Ten percent AS appears at least as good as 10% FCS with regard to both isolation and expansion of human MSC, while 1% and 3% AS appear inferior. With respect to osteogenic differentiation, 10% AS proved superior to the other serum conditions.

Adipocytes↗

PubMed2004

Identification of the adult human hemangioblast.

Recent studies show that human CD133(+) (previously known as AC133(+)) cells from mobilized peripheral blood consist of stem cells with either hematopoietic or endothelial potential. To test whether this population also contains individual precursors with both capacities, the defining characteristics of the elusive adult hemangioblast, we developed a culture system that allows single-cell analyses of differentiation. In the presence of vascular endothelial growth factor (VEGF), stem cell growth factor (SCGF), and FLT-3 ligand, CD133(+)-enriched cells were first expanded and the amplified cells were transduced with a vector encoding an enhanced green fluorescent protein (EGFP) marker gene. Single EGFP(+) cells were then cocultured with corresponding non-transduced cells from the same experiment, yielding 50-100 marked cells in 8% of the wells after 2 weeks. The resultant cells were divided and differentiated with either granulocyte colony-stimulating factor (G-CSF) or with SCGF and VEGF. These culture conditions resulted in the formation of neutrophil or endothelial cells, respectively, as identified morphologically and by phenotypic staining. Dual differentiation of EGFP(+) cells could be observed in one-quarter of clones from single-seeded cells, suggesting that 2% of EGFP(+) cells were in fact human hemangioblasts. These cells could be expanded for at least 28 days without losing this dual capacity. Hence, this culture system may be of clinical relevance in the development of cellular therapies for disorders involving hematopoiesis and the vascular system. In addition, our results provide important information related to the development of the vasculature and the potential role of hemangioblasts in vasculogenesis in adult humans.

AC133 Antigen↗

PubMed2004

Kinetics of G-CSF and CD34+ cell mobilization after once or twice daily stimulation with rHu granulocyte-stimulating factor (lenograstim) in healthy volunteers: an intraindividual crossover study.

BACKGROUND: G-CSF given as split dose increased the harvested number of CD34+ cells in comparison to a once daily schedule, but the mechanism is poorly understood. STUDY DESIGN AND METHODS: To investigate the schedule dependency of G-CSF in healthy volunteers with respect to CD34+ cell mobilization, the same dose of G-CSF was applied in four healthy volunteers in two different schedules (once daily vs. split doses twice daily) in a crossover design after a washout period of 3 months. CD34+ cell kinetics in serum were determined as well as G-CSF serum kinetics on Days 1 and 4 after stimulation. RESULTS: In all volunteers, the twice daily schedule led to a higher CD34+ cell count after 4 days of G-CSF stimulation (median, 94.5 vs. 47/ microL; p = 0.05). On Days 1 and 4, there was a higher peak serum concentration of G-CSF serum level after the once daily application (15,175 vs. 6,859 pg/mL and 7440 vs. 2388 pg/mL, respectively) than after the twice daily schedule. In contrast, after the once daily application the minimum serum level of G-CSF serum level was lower than after the twice daily schedule (663 vs. 1361 pg/mL and 246 vs. 441 pg/mL, respectively). No difference of area under the curve for G-CSF was observed on Days 1 and 4 after G-CSF stimulation. CONCLUSION: It is suggested that application of G-CSF twice daily leads to a higher CD34+ cell mobilization owing to a higher minimum serum level and therefore to a more continuous serum baseline level resulting in a more efficient CD34+ cell mobilization.

Adjuvants, Immunologic↗

PubMed2004

Detection of virulence-associated genes not useful for discriminating between invasive and commensal Staphylococcus epidermidis strains from a bone marrow transplant unit.

Because of their biofilm-forming capacity, invasive Staphylococcus epidermidis isolates, which cause the majority of nosocomial catheter-related bloodstream infections (BSIs), are thought to be selected at the time of catheter insertion from a population of less virulent commensal strains. This fact allows the prediction that invasive and contaminating strains can be differentiated via detection of virulence-associated genes. However, the hospital environment may pave the way for catheter-related infections by promoting a shift in the commensal bacterial population toward strains with enhanced virulence. The distribution of virulence-associated genes (icaADBC, aap, atlE, bhp, fbe, embp, mecA, IS256, and IS257), polysaccharide intercellular adhesin synthesis, and biofilm formation were investigated in S. epidermidis strains from independent episodes of catheter-related BSIs in individuals who have received bone marrow transplantation (BMT). The results were compared with those obtained for commensal S. epidermidis isolates from hospitalized patients after BMT and from healthy individuals, respectively. The clonal relationships of the strains were investigated by pulsed-field gel electrophoresis. icaADBC, mecA, and IS256 were significantly more prevalent in BSI isolates than in commensal isolates from healthy individuals. However, the prevalence of any of the genes in clonally independent, endogenous commensal strains from BMT patients did not differ from that in invasive BSI strains. icaADBC and methicillin resistance, factors important for the establishment of catheter-related infections, already ensure survival of the organisms in their physiological habitat in the hospital environment, resulting in a higher probability of contamination of indwelling medical devices with virulent S. epidermidis strains. The dynamics of S. epidermidis populations reveal that detection of icaADBC and mecA is not suitable for discriminating invasive from contaminating S. epidermidis strains.

Adolescent↗

PubMed2003

Dose finding with retroviral vectors: correlation of retroviral vector copy numbers in single cells with gene transfer efficiency in a cell population.

Retroviral vectors are commonly used in clinical gene therapy, but recent observations of insertional oncogene activation in preclinical and clinical settings have forced a discussion of their safety. Here we investigated the relationship between retroviral transduction efficiency in mass cultures and the actual number of integrated vector copies in single cells using K562 leukemia and primary CD34+ cells. We found an exponential increase of integration numbers correlated to gene transfer rates and a linear increase of expression levels with insertion frequency. On average we detected one vector insertion per transduced cell for a gene transfer of less than 30%, 3 for 60%, and approximately 9 for 90% (in K562). Clonal analysis revealed strikingly increased variations of both transgene copy numbers (more than 20-fold in primary cells) and expression levels associated with higher transduction. Therefore, limiting retroviral gene transfer to approximately 30% may be suggested to avoid generating clones containing multiple insertions.

Cell Culture Techniques↗

PubMed2003

Hepatic lineages isolated from developing rat liver show different ways of maturation.

Immunocytochemical analysis revealed that different hepatic cell types exist during liver development: (i). cells co-expressing the stem-cell marker Thy1 and the hepatic lineage marker CK-18 and (ii). cells only expressing CK-18 (hepatoblasts). In this study we separated the different hepatic cells and analyzed gene-expression and phenotype. Fetal rat livers were digested by collagenase solution. OX43- and OX44-positive hematopoietic cells were depleted and Thy1-positive cells were enriched using Magnetic cell sorting. The different cell compartments were analyzed by RT-PCR and immunocytochemistry for Thy1, CK-18, AFP, and albumin. Hepatoblasts expressed albumin at all times and AFP in the early stages. Thy1-enriched cells expressed CK-18 at all times, albumin in the early, and AFP in the late stages. Thy1-positive cells from fetal livers express liver specific genes. The data suggest that Thy1-positive hepatic cells develop towards hepatic stem cells, and hepatoblasts develop towards mature hepatocytes of the adult liver.

Albumins↗

PubMed2003

Characterization of cell types during rat liver development.

Hepatic stem cells have been identified in adult liver. Recently, the origin of hepatic progenitors and hepatocytes from bone marrow was demonstrated. Hematopoietic and hepatic stem cells share the markers CD 34, c-kit, and Thy1. Little is known about liver stem cells during liver development. In this study, we investigated the potential stem cell marker Thy1 and hepatocytic marker CK-18 during liver development to identify putative fetal liver stem cell candidates. Livers were harvested from embryonic and fetal day (ED) 16, ED 18, ED 20, and neonatal ED 22 stage rat fetuses from Sprague-Dawley rats. Fetal livers were digested by collagenase-DNAse solution and purified by percoll centrifugation. Magnetic cell sorting (MACS) depletion of fetal liver cells was performed using OX43 and OX44 antibodies. Cells were characterized by immunocytochemistry for Thy1, CK-18, and proliferating cell antigen Ki-67 and double labeling for Thy1 and CK-18. Thy1 expression was found at all stages of liver development before and after MACS in immunocytochemistry. Thy1 positive cells were enriched after MACS only in early developmental stages. An enrichment of CK-18 positive cells was found after MACS at all developmental stages. Cells coexpressing Thy1 and CK-18 were identified by double labeling of fetal liver cell isolates. In conclusion, hepatic progenitor cells (CK-18 positive) in fetal rat liver express Thy1. Other progenitors express only CK-18. This indicates the coexistence of different hepatic cell compartments. Isolation and further characterization of such cells is needed to demonstrate their biologic properties.

Animals↗

PubMed2003

Influence of anti-thymocyte globulin as part of the conditioning regimen on immune reconstitution following matched related bone marrow transplantation.

The aim of this work was to analyze the influence of anti-thymocyte globulin (ATG) as part of the conditioning regimen on immune reconstitution following matched related bone marrow transplantation. The rate and pattern of the recovery of total lymphocytes, natural killer (NK) cells, and several T and B cell subsets were determined in 38 patients for more than 2 years following BMT. We compared two patient groups: the first comprised 19 patients after matched related BMT without ATG prevention for graft-versus-host disease (GVHD) and the second contained 19 patients after matched related BMT with ATG treatment for GVHD prophylaxis. We observed impaired immune reconstitution in the ATG group in comparison with the non-ATG group, indicating a significant influence of ATG on immune recovery for several months after BMT.

Adolescent↗

PubMed2003

Effective protection of allogeneic stem cell recipients against Aspergillosis by HEPA air filtration during a period of construction--a prospective survey.

High incidence of aspergillosis on transplant units or hematological wards without HEPA air conditioning during periods of demolishing or construction has been reported by several investigators. Here we report monitoring of fungal air contamination during a period of construction on a stem cell transplantation ward using the gravity air-setting plate (GASP) method. Fungal air contamination in HEPA-conditioned patient rooms was constantly low, independent from construction activity. Outside of the patient rooms at the ward's corridor, the fungal load was significantly higher with some peak values. Outside the transplant unit measures of construction led to a significant increase of fungal spore concentration in air. Transplant activity was not reduced during construction and patients were nursed strictly under HEPA conditions. Patients were monitored prospectively for incidence of infections since 1990 and data of patients grafted during construction (n = 28) were compared to those grafted outside building activity (n = 652). An increase of aspergillosis during construction could be clearly excluded. It can be concluded: Nursing of patients undergoing stem cell transplantation in HEPA-conditioned rooms is an effective protection against acquisition of aspergillus-infection, even under environmental conditions with increased air contamination by conidia. The gravity air-setting plate (GASP) method is not expensive and easy to use and allows reliable and quantitative aerobiological spore monitoring.

Air↗

PubMed2003

Liver-specific gene expression in cultured human hematopoietic stem cells.

Hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. Based on the recent observations that hepatocytes may originate from bone marrow, we investigated the potential of CD34(+) bone marrow cells to differentiate into hepatocytic cells in vitro. CD34(+) and CD34(-) human bone marrow cells were separated by magnetic cell sorting. Cells were cultured on a collagen matrix in a defined medium containing hepatocyte growth factor. Cell count and size were measured by flow cytometry, and reverse transcription polymerase chain reaction was carried out for the liver-specific markers CK-19 and albumin. During cell culture, CD34(+) cells showed an increasing cell number and proliferative activity as assessed by Ki-67 staining. Under the specified culture conditions, CD34(+) cells expressed albumin RNA and CK-19 RNA after 28 days, whereas CD34(-) cells did not show liver-specific gene expression. The results indicate that CD34(+) adult human bone marrow stem cells can differentiate into hepatocytic cells in vitro.

Adult↗

PubMed2002

Genetic protection of repopulating hematopoietic cells with an improved MDR1-retrovirus allows administration of intensified chemotherapy following stem cell transplantation in mice.

This study was undertaken to analyze the hematotoxicity of paclitaxel (Taxol) and to test whether transduction of repopulating hematopoietic cells with a retroviral vector (SF1m) expressing the human multidrug resistance 1 gene (MDR1) would permit dose intensification following bone marrow transplantation (BMT). While the regimen chosen (8 x 20 mg/kg i.p. within 12 days) produced a non-lethal, reversible hematotoxicity in mice with steady-state hematopoiesis, only 35.3% (6/17) of control mice survived when treated starting 14 days post BMT. In contrast, 83.3% (15/18) of mice transplanted with SF1m-transduced cells survived, owing to a significant protection against severe acute myelotoxicity (as determined by neutrophil counts, white and red blood cell counts and values for hemoglobin and hematocrit). After recovery from chemotherapy, an increase of myeloid cells that were resistant to colchicine and effluxed the fluorochrome Rhodamine 123 was observed in SF1m-mice, but not in controls. These results reveal that the lethal, dose-limiting hematotoxicity of an intensified post-transplantation chemotherapy with paclitaxel can be prevented by retroviral transfer of the MDR1 gene to a minor proportion of repopulating cells. Our mouse model, mimicking clinically achievable gene transfer rates, thus suggests that bone marrow chemoprotection may widen the therapeutic window and permit an earlier onset of post-transplantation chemotherapy.

Animals↗

PubMed2002

Highly efficient retroviral gene transfer based on centrifugation-mediated vector preloading of tissue culture vessels.

Efficient retroviral gene transfer into primary cells is a prerequisite for various gene therapeutic strategies. We have developed a transduction protocol based on the preloading of tissue culture vessels with retroviral particles by low-speed (1000g) centrifugation. We show that vector-preloaded tissue culture vessels allow highly efficient gene transfer into various target cells. We obtained transduction rates of up to 85% for primary T lymphocytes after just a single round of transduction. Under clinically relevant conditions using a vector developed for suicide gene therapy and produced under good manufacturing practice (GMP) conditions, the described method allowed generation of large numbers (>2x10(9)) of gene-modified T cells. The preloading concept ensures transduction of target cells in their optimal growth medium regardless of the medium used for vector production. This facilitated highly efficient gene transfer into quite different target cells such as CD34(+) and AC133(+) bone marrow progenitor as well as mesenchymal stem cells. The presented method combines high gene-transfer rates with a great potential for standardization in accordance with GMP guidelines and is consequently well suited for both research and clinical applications. (c)2002 Elsevier Science (USA).

Cell Culture Techniques↗

PubMed2002

Ex vivo expansion of megakaryocyte progenitors from cryopreserved umbilical cord blood. A potential source of megakaryocytes for transplantation.

OBJECTIVE: Umbilical cord blood (CB) provides an alternative source of hematopoietic progenitor cells for transplantation; however, prolonged thrombocytopenia remains a major obstacle due to the low numbers of megakaryocyte progenitor (Mk-prog) cells and their subsequent delayed engraftment. In this study, we improved techniques for enrichment, cryopreservation, and ex vivo expansion of Mk-prog cells from CB. MATERIALS AND METHODS: CB mononuclear cells (MNC) were isolated and Mk-prog enriched by sedimentation on gelatin followed by centrifugation with Ficoll-Hypaque and cryopreserved. The capacity of MNC to produce Mk-prog cells, assessment of CD34(+) and Mk-prog expansion in liquid culture, and analysis of the cell populations by flow cytometry were studied in cryopreserved separated CB and compared to whole CB and freshly separated samples. RESULTS: Excellent viability of greater than 85% was maintained after cryopreservation of separated CB. The number of colony-forming Mk-prog, myeloid, and erythroid progenitor cells did not decrease with cryopreservation. Flow cytometric analysis of cryopreserved cells revealed significant removal of the residual red blood cells while maintaining complete recovery of CD34(+), CD41(+) (Mk), myeloid, and T and B cells compared to noncryopreserved CB cells. There was no difference in the ability of separated cryopreserved MNC CB cells to be expanded in short-term liquid cultures. CONCLUSIONS: The conditions defined here for cryopreservation of gelatin/Ficoll-Hypaque separated CB, followed by ex vivo expansion of MNC, allowed complete recovery of proliferating CD41(+), CD34(+), Mk-prog cells, and other hematopoietic progenitors. Mk-prog cell expansion just before the scheduled transplantation is easily applicable by this technically simple and economical procedure that requires only an aliquot of red cell cell-depleted MNC to be separated from the CB unit before cryopreservation.

Blood Preservation↗

PubMed2002

Allogeneic stem cell transplantation of adult chronic myelomonocytic leukaemia. A report on behalf of the Chronic Leukaemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT).

We report the results of 50 allogeneic transplantations from related (n = 43) or unrelated (n = 7) donors, performed for chronic myelomonocytic leukaemia (CMML) in 43 European centres. The median age at transplant was 44 years (range 19-61). Eighteen patients had excess blasts ranging from 5% to 30% at the time of transplantation. Two graft failures were observed (4%). Neutrophil (> 0.5 x 109/l) and platelet engraftment (> 50 x 109/l) was reached after a median of 17 d (range 11-38) and 27 d (range 11-48) respectively. Acute graft-versus-host disease (GvHD grade II-IV was seen in 35% of patients, while 20% developed severe-acute GvHD grade III/IV. Twenty-six patients (52%) died of treatment-related causes. After a median follow-up of 40 months (range 11-110), the 5-year-estimated overall survival was 21% (95% CI: 15-27%) and the 5-year-estimated disease-free survival (DFS) was 18% (95% CI: 13-23%). Earlier transplantation in the course of disease, male donor, use of unmanipulated grafts, allogeneic transplantation and occurrence of acute GvHD favoured better DFS, but did not reach statistical significance. The 5-year estimated probability of relapse was 49%. The data showed a trend for a lower relapse probability of acute GvHD grade II-IV (24% vs 54%; P = 0.07), and for a higher relapse rate in patients with T cell-depleted grafts (62% vs 45%), suggesting a 'graft-versus-CMML effect'.

Acute Disease↗

PubMed2002

Dose and schedule effect of G-GSF for stem cell mobilization in healthy donors for allogeneic transplantation.

In the present review, we analyze the literature regarding the dose and schedule effects of granulocyte stimulating factor (G-CSF) for stem cell mobilization of healthy donors for allogeneic stem cell transplantation. There is now evidence for a dose and schedule dependency of G-CSF in mobilizing peripheral blood progenitor cells (PBSC) in healthy donors for allogeneic stem cell transplantation. In general, a dose between 10 and 16 microg/kg split into two doses is recommended. Leukapheresis should be performed on day 4 or 5. A higher dose of G-CSF might be appropriate in donors with low CD34+ baseline cell count (< 2000/ml) or if a high CD34+ cell number is required. However, a higher dose of G-CSF results in a higher acute toxicity like bone and muscle pain or headache. Severe adverse events like thromboembolic events, cerebrovascular incidents, anaphylactoid reactions and an atraumatic splenic rupture have been rarely reported. A prolonged follow-up of the donors is needed to rule out late toxicity of the donors.

Blood Donors↗

PubMed2002

Human mesenchymal stem cells are not of donor origin in patients with severe aplastic anemia who underwent sex-mismatched allogeneic bone marrow transplant.

Stromal defects are part of the etiology of severe aplastic anemia (SAA), and hematopoietic engraftment is poor in unrelated and mismatched transplant. Therefore, we wanted to find out whether human mesenchymal stem cells (MSC) are partly of donor origin in patients with SAA years after successful bone marrow transplant (BMT). Three SAA patients 3, 5, and 8 years after BMT (cyclophosphamide, ATG) with bone marrow from an HLA-identical sibling donor of the opposite sex were investigated. MSC were grown from patients' bone marrow aspirates according to Caplan et al. The number of MSC that were isolated from SAA bone marrow post transplant was about 10 times lower than in normal controls. Primary cultures of adherent MSC and passage-one cells were analyzed by dual-color interphase fluorescence in situ hybridization (FISH) analysis using centromere-specific DNA probes for X and Y chromosome. FISH did not show any clear evidence of donor cells in the adherent MSC: In all cases, less than 0.5% of nuclei showed a donor-type signal pattern that is well within assay limits. In a female patient, the absence of male donor cells was confirmed by sensitive and quantitative, Y chromosome-specific TaqMan PCR (QYCS-PCR). In contrast, Ficoll-separated hematopoietic cells from the same aspirates were greater than 90% of donor origin, as expected. In SAA, as previously found in patients with lysosomal and peroxisomal storage disease, bone marrow MSC remain host-derived despite successful hematopoietic engraftment years after allogeneic BMT.

Adult↗

PubMed2002

Allogeneic transplantation after reduced conditioning in high risk patients is complicated by a high incidence of acute and chronic graft-versus-host disease.

Background and Objectives. We studied the toxicity and efficacy of reduced intensity conditioning followed by allogeneic stem cell transplantation in 50 patients over 50 years old or with relative contraindications against myeloablative regimens. Diagnoses were chronic myeloid leukemia (n=15), acute myeloid leukemia (n=9), myelodysplastic syndromes (n=9), lymphoma (n=11) and refractory solid tumors (n=6). Design and Methods. Donors were identical siblings (n=25), non-identical family members (n=6) and unrelated volunteers (n=19). Peripheral blood stem cells (n=36) or bone marrow (n=14) were transplanted. The conditioning regimen consisted of fludarabine 180 mg/m(2), busulphan 8 mg/kg and rabbit antithymocyte globulin 40 mg/kg (Fresenius). Graft-versus-host disease (GVHD) prophylaxis was carried out with cyclosporin A (CSA) alone (n=17) or in combination with methotrexate (n=18) or mycophenolate mofetil (n=15). Results. Neutrophil counts >0.5/nL and platelet counts > 20/nL were reached after 17 (range 0-66) and 19 days (range 0-111), respectively. Three graft failures occurred. Fever lasted for a median of 2 days (range 0-15). Six patients developed veno-occlusive disease of the liver. Acute GVHD grade II-IV occurred in 47% of the patients and chronic GVHD in 46%. The 1-year overall survival probability was 44% (95% CI: 30-58%). GVHD-related complications were a major cause of the probability of 1-year non-relapse mortality of 31% (95% CI: 16-46%). Interpretation and Conclusions. In conclusion, the regimen itself can be carried out safely in patients with relative contraindications against myeloablative conditioning. However, GVHD causes significant non-relapse mortality in high risk patients.

Acute Disease↗