bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Endothelial Cells Generated In Vitro from Circulating Progenitors Represent a Safe Tool for Targeting of Myocardial Tissue.
Authors: Koeck Ruth , Wolf Dominik G.F. , Rumpold Holger F. , Ott Harald , Untergasser Gerold , Erdel Martin , Debagge Paul , Gastl Guenther A. , Gunslius Eberhard
Published: April 2020

Various cell sources, culture protocols and characerization steps are being used in the research field of endothelial progenitors and circulating endothelial cells. We generated EPC by plating the mononuclear fraction onto a collagen I-coated well of a six-well-plate in endothelial cell medium. After 6 to 12 days, 1–10 clones with cobblestone morphology appeared and grew rapidly to confluency within a few days. The rapid proliferation of EPC decelerated after 30–40 days, remaining on the same level for additional 30 days and finally stopped after 70–80 days of culture. Immunofluorescence staining and FACS analysis revealed an endothelial phenotype: EC stained positive for vWF, CD31, CD34, KDR, CD105, CD146, bound UEA-1 lectin and took up acetylated LDL. Of note, CD14 and CD45, which are surrogate markers of monocytes and leukocytes, respectively were negative. The expression of ICAM-1 was upregulated by incubation with TNF-a. In addition, EC formed tubes on Matrigel and expressed VEGF-R1 as well as VEGF-R2-mRNA proven by RT-PCR. To gain deeper insight into the regulation of cellular senescence, we performed western blot analyses of cell cycle regulating proteins. In contrast to early passage cells, growth arrested cells from late passages up-regulated p15, p16, p21, whereas p27 and Id1 were significantly dowregulated. Moreover, late-passage cells highly expressed SA-ß-galactosidase. The obvious replicative senescence together with the regular karyogram indicates a relative safety aspect of their application in vivo. To study their homing behaviour to ischemic tissue, we established a model of myocardial infarction, which was induced by ligation of the left anterior descending coronary artery in Rowett nude rats. 5–10x106 PKH red-labeled EPC were injected into the tail vein. Intramyocardial injection served as positive control, while administation of EPC without prior infarction represented the negative control. Rats were sacrificed on day 14 after the operation and cryosections of their hearts were prepared. PKH-positive cells were almost exclusively found in the infarcted left ventricle after i.v.-application, thus showing a high specifity for ischemic myocardium. The results were confirmed by immunohistochemical staining of human CD31.

In summary, EPC generated according to the described protocol represent a possible tool for safe cell-based therapy strategies.
Ex-Vivo Expansion of Multipotent CD133+ Stem Cells with VEGF, FLT3l and SCGF.
Authors: Loges Sonja , Butzal Martin , Fischer Uta , Gehling Ursula M. , Hossfeld Dieter K. , Fiedler Walter
Published: April 2020

The rare CD133+ stem cell population contains both hematopoietic and endothelial progenitors. Successful ex-vivo expansion of this multipotent population would therefore be of great benefit in many clinical settings including stem cell transplantation and gene therapy. We developed a cell culture system containing the recombinant human cytokines vascular endothelial growth factor (VEGF), FLT3 ligand (FLT3L) and stem cell growth factor (SCGF) for ex-vivo expansion of purified human CD133+ stem cells obtained from leukapheresis products from patients pre-treated with G-CSF.

FACS analysis, colony assays and NOD-SCID transplantation studies were performed to monitor stem cell and endothelial phenotype in-vitro and in-vivo. Cultivation with VEGF, FLT3L and SCGF induced a mean 2200-fold increase of total cell counts in 5 weeks. FACS analysis revealed persistence of 6–15% CD133+ stem cells indicating proliferation and survival of primitive hematopoietic stem cells. 5–6% of the proliferating cells expressed the endothelial markers CD144 (VE-Cadherin) and von-Willebrand factor (vWF). Ex-vivo expanded stem cells could be differentiated into adherent endothelial cells after withdrawal of SCGF and FLT3L allowing generation of large numbers of endothelial cells. Colony-assays showed an increase of hematopoietic and endothelial colonies after 5 weeks of ex-vivo expansion indicating simultaneous proliferation of hematopoietic and endothelial precursors under the established culture conditions (CFU-E 60-fold, CFU-GEMM 48-fold, CFU-GM 59-fold, CFU-G 99-fold, CFU-M 1356-fold and CFU-EC 1843-fold).

To assess in-vivo functionality, hematopoietic stem cells expanded ex-vivo for 7, 14, 21 and 32 days were transplanted into sublethally irradiated NOD-SCID mice. For each expansion period, the mean percentage of anti-human CD45 positive bone marrow cells 3 months post-transplantation was 11, 3, 3 and 1%, respectively. Human CD45+ cells for each set of experiments contained a mean of 15, 26, 8 and 32% T-cells (CD3+), 9, 0, 7 and 21% B-cells (CD19+), 24, 2, 2 and 11% monocytes (CD14+), 21, 3, 1 and 12% granulocytes (CD33+) and 19, 37, 44 and 24% stem cells (CD34+) (d7 (n=5), d14 (n=4), d21 (n=7) and d32 (n=6) respectively).

Our experiments showed multilineage engraftment of human stem cells expanded for more than 4 weeks ex-vivo. Therefore our culture system provides a tool to generate large numbers of human stem and endothelial cells for clinical purposes.
HOXB4-Mediated Immortalization of Murine Hematopoietic Stem/Progenitor Cells.
Authors: Zhang Xiao-Bing , Kiem Hans-Peter
Published: April 2020

HOXB4 overexpression has been shown to be able to expand hematopoietic stem/progenitor cells (HSPCs) in short-term ex vivo culture. Here we found that HOXB4-transduced cells could expand continuously in up-to-eight-month long-term liquid culture. In 3 independent experiments, mouse bone marrow (BM) Sca1+ cells were transduced with HOXB4 or YFP vectors and kept culture in IMDM/10%FCS containing SCF, Flt3-L, and TPO each at 50 ng/ml. After 3–4 weeks of culture, all YFP-expressing cells differentiated and died. However, HOXB4-transduced cells continuously expanded and percentage of HOXB4-expressing cells increased to more than 95% after 4 weeks of culture. Without cytokine support, these cells stopped growing, while a single cytokine, SCF, was able to support the expansion of these cells, albeit at a lower rate. In the presence of SCF, TPO and Flt3-L, these cells expanded robustly with cell doubling time of around 2 days. These cells are morphologically immature by cytospin and Wright-Giemsa staining. CFU assay showed a clonogenicity of 2%. Phenotyping by FACS showed that all the cells expressed c-kit, while some cells expressed Gr-1, Mac-1 and CD3, indicating that HOXB4-immortalized cells spontaneously differentiate into myeloid and lymphoid cells in ex vivo culture. To test the repopulating capability of these cells, 1x107 cells were injected into each sublethally irradiated NOD/SCID mice via tail vein. These cells engrafted in the recipients with 2% in BM and 10% in spleen. Transplantation into lethally irradiated congenic CD45.1 mice is in progress. Immortalization of human CD34+ by HOXB4, however, failed. To determine the mechanisms under which HOXB overexpression immortalizes mouse rather than human HSPCs, we performed Western blot assay with HOXB4-specific antibody. HOXB4 protein levels in human cells decreased continuously from 1–3 weeks posttransduction, whereas expression in mouse cells remained stable. Furthermore, we found that HOXB4 transduced in mouse cells was tyrosine phosphorylated, which is evidenced by treatment of cells with sodium orthovanadate, a protein tyrosine phosphatase inhibitor, treatment of cell extracts with l-phosphatase and immunoprecipitation with phosphorylated tyrosine-specific antibody. Consistent with other reports, no leukemogenesis was observed in our studies as yet. It would be interesting to compare the differences in gene expression profiles between the HOXB4- and HOXA9-immortalized hematopoietic cells, which may unveil the molecular mechanisms governing leukemogenesis. In conclusion, murine HSPCs can be immortalized by HOXB4 overexpression and tyrosine phosphorylation of HOXB4 may be responsible for the self-renewal and immortalization of HSPCs.
The Tetraspanin CD82 Defines Erythroid Committment, Regulates Stem- and Progenitor Cell Survival and Mediates Adhesion.
Authors: Burchert Andreas , Denecke Barbara , Haerle Tobias , Neubauer Andreas
Published: April 2020

CD82 belongs to the tetraspanin superfamily, members of which regulate multiple aspects of cell biology such as T-cell activation, proliferation, differentiation, and adhesion.We have previously shown expression of CD82 on hematopoietic progenitor cells. Here we were seeking to shed light on the functional role of CD82 in hematopoiesis. First, we found that CD82 expression is strongly associated with an erythroid committment as exclusively the CD82bright fraction but not the CD82medium or CD82dim fraction of CD34+ cells gave rise to erythroid colony forming units (CFU). In order to manipulate CD82 activity, we used as surrogate ligand, a previously described, CD82-activating monoclonal antibody (moAb), clone 50F11, to activate CD82 on hematopoietic precursors. We found that 50F11, but not another CD82 specific antibody clone, BL-2, specifically induced tyrosine phosphorylation of CD34+ cells. In Dexter-type long-term cultures (D-LTC) 50F11, and not IgG1 isotype control moAbs significantly inhibited myelopoiesis and the number of CD34+ clonogenic progenitors. Moreover, in long term culture initiating cell (LTC-IC) assays, 50F11as compared to isotype control antibodies substantially inhibited, but not entirely abolished, the number of 5 week-LTC-IC′s, indicating that CD82 activation inhibits progenitor - and stem cell proliferation or self renewal. Finally, plastic immobilized 50F11-antibodies caused a time-, and concentration dependent induction of adhesion of CD34+ cells, which was associated with the formation of F-actin and development of multipolar extensions. Finally, CD82 ligation by 50F11 caused a statistically significant down-regulation of the integrin CD49d (p=0,036) and CD62L (p=0,010).

Together, it is shown that CD34+/CD82high cells characterize an erythroid committment implying a role for this tetraspanin in erythroid hematopoiesis. Activation of CD82 induces adhesion and negatively regulates proliferation of adult stem- and progenitor cells. This implicates a so far unknown role for CD82 in the regulation of early hematopoiesis.
Intracardiac Transplantation of Already Specifically Dedifferentiated Circulating CD34+ Cell Subsets Would Favor Post-Ischemic Myocardial Regeneration.
Authors: Henon Philippe R.G. , Sovalat Hanna , Ojeda-Uribe Mario , Arkam Yazid , Bischoff Nicolas , Monassier Jean-Pierre , Dallemand Robert
Published: April 2020

Several groups, mainly from Germany and Japan, have recently conducted different phase-I clinical studies in which autologous bone marrow (BM) mononuclear cells (MNCs) were reinjected either directly in the ischemic area or intra-coronary in patients with severe post-infarct cardiac failure, resulting in a significant improvement of myocardium viability and/or reperfusion. However, besides “true” HSCs, BM-MNCs represent a mixture of mesenchymal progenitor cells, angioblasts, and maybe other progenitor cells, which does not allow to identify the type(s) of cells potentially responsible for improvement. Moreover, the obstructed coronary artery was always repermeabilized, which biases the evaluation of posttransplant myocardial reperfusion. We have personally chosen an other original approach using mobilized and purified circulating CD34+ cells. We and others have indeed demonstrated that mobilized CD34+cells were in fact subdivided into various subsets: of course, the most important (~75%) is the truly hematopoietic subset (CD34+/133+), of which a CD38− part is probably close to the very primitive HSC. But other smaller subsets are immunophenotypically characterized either as mature (CD34+/VEGFR-2+) or immature (CD34+/133+/VEGFR-2+) endothelial progenitor cells - thus potentially capable of neoangiogenesis -, or as muscle progenitors (Desmin+) and even more as cardiomyocytes (Troponin-T+). In a phase-I trial benefiting of the approval of the regional ethical committee, patients suffering of post-infarct cardiac failure are selected according to the following criteria: left ventricular ejection-fraction (LVEF) =35%; distinct area of left ventricular-wall akinesis determined by PetScan; candidates for coronary artery by-pass grafting (CABG), but without any repermeabilization of the coronary artery involved in the infarction; age =70 y. After a 6-days mobilization by G-CSF, circulating CD34+ cells are collected, then purified by immunomagnetism and immediately reinjected at d+7 during CABG, all around and within the infarcted area. The first evaluable patients well tolerated cell mobilization - and collection phases, as well as operative and post-operative periods. Three patients have presently a follow-up = 1 y post-transplantation. Two show a striking gain in LVEF (14 and 20% respectively) with an important improvement in myocardium viability, reperfusion and contractility, and finally in exercise capacity (from class IV to class I in New-York Association functional class). Although very encouraging, these results have to be confirmed in further patients.
Cord Blood Mesenchymal Stem Cells Are Enriched for Neural and Glial Progenitors.
Authors: El-Badri (Dajani) Nagwa S. , Saporta Samuel , Liang Xiaomei , Sanberg Cyndy D. , Sanberg Paul R.
Published: April 2020

Human bone marrow stroma is the primary source for mesenchymal stem cells (MSCs), a mixture of multipotent progenitors that differentiate into fibrous, cartilage, adipose, bone and reticular tissue cells. Recent reports suggest that human umbilical cord blood (UCB) is a feasible source for MSCs. In this study, we examined whether MSCs from umbilical cord blood contain progenitors that differentiate into cells of neural and glial lineages. UCB mononuclear cell fraction was cultured in Iscove’s MDM culture medium supplemented with 20% fetal bovine serum and L-glutamine. After 2 weeks in culture, adherent cells formed colony forming unit fibroblast (CFU-F)-like colonies. When adherent cells reached 70% confluency, they were further subcultured in the same medium for three more weeks before replacing IMDM with neurogenic differentiation medium. Cord blood MSCs were devoid of hematopoietic activity since they failed to produce CFU-GM, CFU-E, or CFU-GEMM hematopoietic colonies when cultured in methyl cellulose medium supplemented with the appropriate hematopoietic growth factors. Flow cytometric analysis showed a population of cells devoid of hematopoietic phenotype (CD34−, CD45−, CD38−, CD3−, CD19−). After 7 days incubation in serum free supplemented Neurobasal™ medium, cultured cord blood MSCs expressed neuron cell surface antigen A2B5, neurofilament polypeptide NF200, oligodendrocyte marker 04, and intermediate filament proteins, nestin and vimentin. Strong immunoreactivity was observed with astrocyte marker GFAP. A proportion of cultured MSCs developed into neuron-like cells that were positive for TUJ-1 neural progenitor marker. These results indicate that human UCB-derived MSCs are enriched for neuro-glial progenitors, and could be further developed for the treatment of stroke, spinal cord injury, and neurodegenerative diseases such as like Alzheimer’s, Parkinson’s and Multiple Sclerosis.
Epigenetic Regulation of the Dendritic Cell Marker Gene MADDAM /ADAM19.
Authors: Kreutz Marina , Ehrnsperger Achim , Rehli Michael , Andreesen Reinhard
Published: April 2020

MADDAM (Metalloprotease And Disintegrin Dendritic Cell Antigen Marker, ADAM19), a human metalloprotease belonging to the ADAM-family, is strongly expressed during in vitro differentiation of monocytes into dendritic cells (DC), whereas differentiation of monocytes into macrophages (MAC) is associated with a loss of MADDAM transcription. To investigate the mechanisms underlying this cell-type specific expression pattern we defined the transcriptional start site and the proximal promoter of the MADDAM gene. Gene bank analysis of the CpG island promoter and first intron revealed putative binding sites for several transcription factors, including VDR, NF-kB and Sp1-family factors. EMSA demonstrated binding of Sp1, Sp3, NF-kB and VDR to their putative binding sites in the proximal promoter region and mutation of these elements led to a decreased reporter activity of the proximal promoter in luciferase assays. A minimal promoter construct of 150-bp showed weak reporter activity in primary monocyte-derived MAC and a threefold higher activity in monocyte-derived DC, indicating that differential binding of transcription factors contributes to the cell-type specific regulation of MADDAM. Transfection of monocytic THP-1 cells with the 150-bp fragment also resulted in significant reporter activity, despite the lack of endogenous MADDAM expression. Interestingly, Trichostatin A (TSA), a known inhibitor of histone deacetylation, lead to a dose dependent induction of MADDAM mRNA in THP-1 cells. Chromatin immunoprecipitation (ChIP) assays demonstrate increased levels of acetylated histones H3 and H4 in DC as compared to MAC, indicating an important role of histone acetylation in the cell-type specific regulation of the MADDAM gene.
Significant Down-Regulation of BAALC during Lineage Specific Hematopoietic Differentiation.
Authors: Baldus Claudia D. , Komor Martina , Hoelzer Dieter , de la Chapelle Albert , Thiel Eckhard , Hofmann Wolf-Karsten
Published: April 2020

The human gene BAALC (Brain And Acute Leukemia, Cytoplasmic) implicated in normal hematopoiesis and leukemia is a marker of immature hematopoietic progenitor cells (PNAS2001;98:13901). To explore its role in hematopoiesis we investigated the regulation of BAALC during lineage specific differentiation. Semi-quantitative RT-PCR was performed on subsets of in vitro differentiated bone marrow CD34+ cells from healthy individuals using the cytokines G-CSF, M-CSF or EPO. Cultures were harvested on day 4, 8, 12, and 16. In addition, oligonucleotide microarray analyses (HG-U133A, Affymetrix) of in vitro stimulated CD34+ cells were independently carried out using EPO, TPO, G/GM-CSF to induce lineage-specific differentiation. For each of the lineages, cells were harvested at days 4, 7 and 11 for expression profiling. All experiments were done in triplicates for each time point and condition. RT-PCR revealed down-regulation of BAALC and CD34 as early as day 4 in cultures with G-CSF, M-CSF or EPO. In concordance, gene expression profiling showed significant down-regulation of BAALC and CD34 on day 4 of culture (BAALC: EPO p=0.04, G/GM-CSF p=0.17, TPO p=0.03; CD34: EPO p=0.01, G/GM-CSF p=0.003, TPO p=0.01). 275 genes showing a significant correlation (correlation coefficient r>0.95) to BAALC expression levels (at the 4 time points and using 3 different cytokines) were identified. Of these CD34 ranked second (r=0.99), and genes related to leukemia and neuronal cells included: Hepatic leukemia factor (HLF; r=0.99) and Paired box gene 5 (PAX5; r=0.99). It has been speculated that BAALC might be involved in cell movement or cell-cell interaction; we therefore investigated the association to genes implicated in cytoskeletal function. Expression of Myosin 5C (MYO5C; r=0.99) and Synaptic nulcei expressed gene 2 (SYNE2; r=0.95) were highly correlated with BAALC expression across all conditions (EPO, TPO, G/GM-CSF). In conclusion, using two independent approaches we show the significant down-regulation of BAALC during hematopoietic differentiation. This underscores the potential role of BAALC in early hematopoiesis and the possible contribution of aberrant BAALC expression in leukemogenesis. We postulate that similar to CD34, PAX, and HLF implicated in hematopoiesis and leukemia, down-regulation of BAALC and structural genes including Myosin 5C are critical steps during hematopoietic differentiation.
New Role of the Regulatory Gene SOX2 in Hematopoiesis.
Authors: Levantini Elena , Bertolotti Francesca , Cerisoli Francesco , Ferri Anna L. , Brescia Elisa , Galvagno Daniele , Tenen Daniel G. , Ottolenghi Sergio , Nicolis Silvia K. , Magli Maria C.
Published: April 2020

Several genes encoding transcription factors of different families have been implicated in the development and differentiation of multiple cell systems. The Sry-type high-mobility-group box 2 gene (Sox2) encodes a transcription factor that is expressed in very early cells such as embryonic stem cells and neural stem cells, where it plays important functional roles (Genes and Dev.17:126, 2003; Development131:3805, 2004). To investigate whether Sox2 plays a role also in blood cell production, we first analyzed its expression in murine hematopoietic cells. Results indicate that the gene is transcriptionally active at low levels in primitive progenitors. Furthermore, in order to address the functional implication of Sox2 in hematopoiesis we analyzed mature and precursor cells in mutant mice compound heterozygotes for a null Sox2 allele and for the deletion of a Sox2 5′ enhancer, as the complete inactivation of the gene in homozygosis is embryonic lethal. At the peripheral blood level we did not detect significant variations in the mutants. However analysis of bone marrow precursors in clonogenic assays showed that Sox2 knock-down mice exhibited a significant increase in the number of multipotent precursors, as compared to wild type animals. Moreover, bone marrow cells of wild type and mutant mice were analyzed for the expression of a panel of regulatory genes involved in the control of different somatic stem cells. Preliminary evidence suggests that some of these genes are modulated in the mutant cells. These observations support the view that Sox2 plays a role at early stages of blood cell production, providing further evidence that common molecular mechanisms may be involved in the regulation of several different types of multipotent cells.
Association of IL-10 and IL-10 Receptor Gene Polymorphisms and Graft-Versus-Host Disease Following Hematopoietic Cell Transplantation.
Authors: Tseng Li-Hui , Lin Ming-Tseh , Storer Barry , Martin Paul J. , Grogan Bryan , Chen Pei-Jer , Zhao Lue Ping , Hansen John A.
Published: April 2020

Polymorphisms in cytokine genes can influence immune responses and may affect the outcome of hematopoietic cell transplantation (HCT). We have shown that the IL-10/-592*A allele of the recipient is a marker for less severe acute graft-versus-host disease (GVHD) and a lower risk of non-relapse mortality (NRM) after HCT from an HLA-identical sibling (N Engl J Med, 2003). To further test the hypothesis that IL-10 pathway is important in the intensity of acute GVHD, we undertook a study of variation in the IL-10 receptor β gene. A single nucleotide polymorphism (A/G) at cDNA position 238 of the IL-10 receptor β gene (IL-10RB/1304) was genotyped in 953 HCT recipients and their HLA-identical sibling donors. IL-10/-592 and IL-10RB alleles and genotypes were tested for association with GVHD by multivariable analysis. The IL-10/-592*A allele of the recipient and IL-10RB/238*G allele of the donor were significantly associated with a lower risk of acute grades III-IV GVHD (trend p value 0.0008 and 0.02, respectively). None of the 16 cases with a patient IL-10 A/A genotype and donor IL-10RB G/G genotype developed grades III-IV acute GVHD (HR = 0.0 and p value = 0.007), compared to pairs with a patient IL-10 C/C genotype and donor IL-10RB A/A genotype. The hazard ratios were 0.4–0.6 among pairs with a patient IL-10 A/A genotype and donor IL-10RB A/G or A/A genotype and among pairs with a patient IL-10 A/C genotype and donor IL-10RB G/G or A/G genotype. The effect of donor IL-10RB genotype on GVHD was observed only among pairs with a patient IL-10 A/C or A/A genotype (trend p value = 0.005 and 0.06 respectively), but not among pairs with a patient IL-10 C/C genotype (trend p value = 0.82). These data suggest an interaction in the effect of the patient IL-10/-592 and donor IL-10RB/1304 genotypes on GVHD, further supporting the hypothesis that the IL-10 pathway plays an important role in controlling the severity of acute GVHD.
Pegfilgrastim after High-Dose Chemotherapy and Autologous Peripheral Blood Stem Cells Transplantation. A Single-Centre Experience in 15 Patients.
Authors: Soussain Carole , Marec Laure , Abarah Wajed , Allard Christian , Mallek Hassina , Itani Corine
Published: April 2020

The efficacy of Pegfilgrastim (PF) in decreasing the duration of neutropenia has been proved after standard dose of chemotherapy. Results of PF after high dose chemotherapy (HDC) and autologous peripheral blood stem cells transplantation ASCT) are lacking. We studied the efficacy of PG in patients receiving HDC and ASCT for lymphoproliferative disease. Fifteen consecutive patients (8 males; 7 females) were onrolled in the study from September 2003 through March 2004. Median age of the patients was 56 years. Diseases were multiple myeloma in 5 patients, diffuse large cell non-Hodgkin’s lymphoma (NHL) in 3 patients, follicular lymphoma in 4 patients, mantle cell lymphoma in one patient, and primary central nervous system lymphoma PCNSL) in 2 patients. All patients were eligible for HDC and ASCT per institutional criteria. Stem cells were collected with peripheral blood pheresis after high dose cyclophosphamide (7 cases); high dose Ara-c (5 cases); ifosfamide (1 case); CHOP-like chemotherapy (1 case); or in steady state (1 case). All the patients received daily G-CSF (5 to 10 mcg/kg). Three different conditioning regimens were used. Patients with multiple myeloma received high dose melphalan (200 mg/m²), patients with PCNSL received a combination of high dose Thiotepa (750 mg/m²), Busulfan (12 mg/kg), and Cyclophosphamide (120 mg/kg), and patients with NHL received a BEAM chemotherapy. PF was administered as a single subcutaneous injection of 6 mg at day +3 after stem cell infusion, except for one patient whose injection was done on day 4. No adverse event attributable to PF was observed. There were no toxic death on study. All patients engrafted neutrophils and platelets. The median time to neutrophils engraftment (> 500/mm3) was 7 days (range, 4–12). Febrile neutropenia was almost constant (14/15) but never exceed OMS grade 2. Median number of days with IV antibiotics was 7 days (range 5–22). These preliminary data show that a fixed dose of 6 mg of PF given subcutaneously at day +3 after HDC and ASCT is safe and effective. A cost efficacy study is warranted to compare PF and daily dose of standard G-CSF after ASCT.
IL-10 Promoter Gene Polymorphism Associated with the Occurrence of Chronic GVHD and Its Clinical Course during Systemic Immunosuppressive Treatment for Chronic GVHD after Allogeneic Peripheral Blood Stem Cell Transplantation.
Authors: Kim Dong Hwan , Lee Nan Young , Sohn Sang Kyun , Baek Jin Ho , Kim Jong Gwang , Lee Jung Lim , Suh Jang Soo , Lee Kun Soo , Lee Kyu Bo
Published: April 2020

Introduction: The current study attempted to evaluate the association between the IL-10 promoter gene polymorphism and transplant outcomes, including the occurrence of chronic GVHD and its clinical course during systemic immunosuppressive treatment (IST) after allogeneic peripheral blood stem cell transplantation (PBSCT).

Methods: Three single-nucleotide polymorphisms in the proximal region of the IL-10 promoter gene (−1082/−819/−592) were analyzed in 60 recipients of cytokine-mobilized PBSCs from HLA-matched sibling donors, and the transplant outcomes compared in terms of the occurrence of chronic GVHD and its clinical course.

Results: The current study only found two haplotypes (1082*A/819*T/592*A [ATA] and 1082*A/819*C/592*C [ACC]). An increased occurrence of chronic GVHD was noted dependent on the IL-10 haplotype (43% vs 68% vs 96% in ACC/ACC vs ATA/ACC vs ATA/ATA haplotype, p=0.003). In a logistic regression based on a multinomial model, the ATA/ATA homozygote presented a 7-fold increased risk of the development of chronic GVHD compared with the ACC/ACC homozygote. The cumulative incidence of chronic GVHD at 1 year post-transplant was 46±20%, 64±10%, and 82±5% in the ACC/ACC, ATA/ACC, and ATA/ATA groups, respectively (p=0.0266). The recipients without the ACC haplotype had a more frequent history of acute exacerbation of chronic GVHD (p=0.031, odds ratio 6.750), while 72% of the recipients with the ACC haloptype did not experience any history of acute exacerbation after the initiation of systemic IST for chronic GVHD compared to 38% of those without the ACC haplotype (p=0.014, odds ratio 4.375). Plus, the duration of systemic IST was significantly shorter in the recipients without the ATA-haplotype compared to those with the ATA haplotype (339 days versus 1,146 days, p=0.0091). In terms of GVHD-specific survival, the recipients without the ATA haplotype showed a 100% survival compared to 70.5% for the ATA haplotype group (p=0.2825).

Conclusion: The IL-10 promoter gene polymorphism was found to be apparently associated with the occurrence of chronic GVHD and its clinical course during systemic IST for chronic GVHD in an allogeneic PBSCT setting.

Figure Figure Figure Figure
Erythropoietin Signaling in Tumor Cells: Differential Activation of Mitogen Activated Protein Kineses and Their Effect upon Cell Survival.
Authors: Udupa Kodetthoor B. , Bose Chhanda
Published: April 2020

Erythropoietin (EPO) regulates the proliferation and differentiation of erythroid progenitors via its receptor, EPO-R and through various Mitogen activated protein kinase (MAPK) pathways. The role of EPO on other cell types is still unkown. In the present study we have elected to examine this aspect in a transformed pancreatic cell line, AR42J cells. We investigated the activation of two MAPKs, namely extracellular regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) after exposure of AR42J cells to 1 U/ml EPO using the Western blot analysis. We also examined cell viability during EPO exposure by high sensitivity fluoremetric method using CCK8. We found a rapid activation of ERK-1/2 in AR42J cells reaching the maximum of 3.3 fold in 5 min, while it took 30 min for JNK-1/2 to reach the maximum. In the absence of EPO, addition of specific JNK inhibitor, SP600125, reduced cell viability to 50% at a dose of 20 μM while with ERK inhibitor, UO126, cell viability was not reduced even up to 60 μM of the drug. To examine the effect of induction of MAPK by EPO on AR42J cell survival, cells were treated with inhibitors to ERK or JNK 1h prior to EPO addition and the cumulative cell survival were calculated from day 1 through 4. EPO addition to AR42J cells resulted in significantly higher cumulative cell survival of 1.00 ± 0.04 unit absorption compared to the value of 0.35 ± 0.02 unit absorption seen in controls without EPO. When cells were treated with EPO and ERK inhibitor a significantly higher cumulative cell survival of 1.50 ± 0.04 unit absorption was observed (p < 0.01) indicating ERK to be less effective in their survival. On the other hand, samples treated with EPO and JNK inhibitor had significantly lower cell survival (0.55 ± 0.06 unit absorption, p < 0.01) than the EPO control indicating an essential role of JNK in their survival. These results indicate that in EPO mediated survival of AR42J cells, activation of JNK appears to be more important than ERK activation thus indicating a key role of JNK in cell viability. It is very important to understand the role of EPO signaling in the viability of transformed cells since EPO is often supplemented after bone marrow transplantation to enhance the recovery after chemotherapy.
Proliferation Versus Differentiation: The Effect of Growth Factor Stimulation Detected by HALO.
Authors: Rich Ivan N. , Hall Karren , Bartelmez Stephen H.
Published: April 2020

In order for stem and progenitor cells to initiate the proliferation process to grow in vitro and form colonies of differentiated cells, growth factors are required. Some factors are specific for proliferation, some for differentiation. Others exhibit both a proliferative and differentiation function. To date, the classical colony-forming assay (CFA) has been used to ascertain the effectiveness of growth factors either individually or in combination. However, the CFA cannot differentiate between a proliferation or differentiation factor because the assay itself is a differentiation assay. We have now developed a unique in vitro system which measures the proliferative capacity of cells to respond to different growth factors. Based on the colony-forming assay, HALO (Hematopoietic Assays via Luminescence Output) measures cell proliferation as a function of the concentration of intracellular ATP which drives a luciferin/luciferase reaction. The end-point of the reaction is bioluminescence detected in a plate reader luminometer. Since the assay does not require cells to differentiate into colonies, no manual enumeration is needed. Furthermore, HALO is a rapid, non-subjective and standardized proliferation assay performed in 96-well plates with high-throughput capability. At least 14 different cell populations with differing proliferative potential from 5 different species can be detected simultaneously. For the present study, both mouse and human bone marrow target cells were used. Eight replicate microcultures of 100ul each were performed to investigate the dose-dependent effect of single growth factors on cell proliferation. The results are summarized as follows. Proliferation of mouse and human hematopoietic cells can be detected between 1 and 2 days and is optimal between 4 and 7 days respectively. The growth factors, SCF, IL-3, IL-6, Flt3-ligand, EPO, TPO and GM-CSF alone, all induce proliferation at less than 1ng/ml. At growth factor concentrations typically used in the colony-forming differentiation assay, many are inhibitory. The EPO dose response for murine CFU-E proliferation measured at 21h produced a flat response and inhibition at doses above 10mU/ml. The mirror image profile was obtained for CFU-E differentiation at 48 h. These results demonstrate that at the CFU-E level, EPO is a differentiation factor and not a proliferation factor. However, EPO alone does stimulate proliferation at the BFU-E stage of development. In conclusion, it is now possible to distinguish between a proliferation and differentiation factor at different hierarchical levels in the hematopoietic system.
Myeloid Progenitor Cells from Gadd45a and Gadd45b-Deficient Mice Are Sensitized to Genotoxic-Stress Induced Apoptosis.
Authors: Gupta Mamta , Gupta Shiv K. , Balliet Arthur G. , Hoffman Barbara , Lieberman Dan A.
Published: April 2020

GADD45 (Growth arrest and DNA damge) regulates cell growth following exposure to diverse stimuli. It has been shown that, mice lacking the gadd45a gene exhibit genomic instability and increased carcinogenesis, but the exact role of the gadd45 family genes still remains unclear. In this study we have aimed at determining the effect of gadd45a or gadd45b deficiency on the response of bone marrow derived myeloid cells to genotoxic stress agents by using gadd45a or gadd45b null mice. We have found that myeloid progenitor cells from gadd45a or gadd45b-null mice are more sensitive to ultraviolet-radiation (UV), VP-16 or daunorubicin induced apoptosis. Introduction of wild-type gadd45 into gadd45-deficient bone marrow cells restored the wild-type apoptotic phenotype. In-vitro colony formation following stress responses has shown that bone marrow cells from gadd45a or gadd45b-deficient mice have a decreased ability to form haematopoetic colonies. Gadd45a or gadd45b-deficient bone marrow cells also displayed defective G2/M cell cycle checkpoint following exposure to either UV and V-16 but were still able to undergo G2/M arrest following exposure to daunorubicin, indicating the existence of different G2/M checkpoints in response to these anticancer agents. Taken together these findings identify gadd45a or gadd45b as anti-apoptotic gene(s), and suggests that the absence of gadd45a or gadd45b results in higher susceptibility of haematopoetic cells to UV radiation and certain anticancer drugs.
Tumor-Derived Lactic Acid Modulates Dendritic Cell Activation and Differentiation.
Authors: Kreutz Marina , Gottfried Eva , Kunz-Schughart Leoni , Hoves Sabine , Andreesen Reinhard , Muller-Klieser Wolfgang , Mackensen Andreas
Published: April 2020

The tumor milieu can influence DC differentiation in vivo and in vitro. We analyzed DC differentiation in a 3-dimensional tumor model and propose a new mechanism of DC modulation by the tumor environment. Monocytes were cultured in the presence of IL-4 and GM-CSF within multicellular tumor spheroids (MCTS) generated from urothelial carcinoma (J82, UMUC3) and melanoma cell lines (MelIm, Mel108). Monocytes invaded the tumor spheroids and differentiated into tumor-associated dendritic cells (TADC) that displayed an altered phenotype compared to DC generated without tumor contact. The expression of CD1a was reduced on TADC whereas CD80, CD86 and CD16 were upregulated. In addition, TADC generated within J82-MCS showed a reduced expression of HLA-DR and accordingly the antigen presentation in a mixed lympocyte reaction was decreased. We then analyzed the underlying mechanism. Supernatants of MCTS could partially transfer the suppressive effect. Conditioned media from urothelial carcinoma cell lines contained high levels of M-CSF and IL-6, both cytokines known to modulate DC differentiation. In contrast, melanoma MCTS cocultures produced little M-CSF and IL-6, but further analysis revealed that melanoma cells secreted high levels of lactic acid. Indeed, addition of lactic acid during DC differentiation in vitro induced a phenotype comparable to TADC generated within melanoma MCTS. Inhibition of melanoma cell lactic acid production by the addition of oxamic acid, an inhibitor of lactate dehydrogenase, reverted the TADC phenotype back to normal. We therefore conclude that tumor-derived lactic acid is an important factor modulating the DC phenotype in the tumor environment which may critically contribute to tumor escape mechanisms.
Mesenchymal Stem Cells (MSC) Exhibit Antigen Presenting (APC) and Phagocytic Properties: Implicatins to Bnone Marrow Failure during Inflammation.
Authors: Chan Jennifer L. , Harrison Jonathan S. , Ponzio Nicholas M. , Rameshwar Pranela
Published: April 2020

Mesenchymal stem cells (MSC) mostly surround the vasculature system of bone marrow (BM). MSC have been shown to exhibit immune suppressive properties. Since MSC express MHC Class II antigen, the question is whether these cells can act as APC. To this end, we hypothesize that MSC have the ability to present non-self antigens while acting as immune modulators. These dual roles of MSC prevent exacerbated inflammatory responses in the BM, thereby preventing hematopoietic dysfunction.

A ‘dampened’ immune response in BM during insults by foreign agents could cause protection of the barrier that separates BM cavity with the periphery. The phagocytic role of MSC was shown by confocal microscopy and fluoresbrite plain YG 1.0-micron microspheres. APC property was demonstrated by challenging MSC with C. albicans (pulsed MSC), followed by exposure to CD4+ cells. The latter was obtained by immunoselection from peripheral blood mononuclear cells (PBMC) cultured for 5 days with C. albicans (10 mg/ml). Proliferation of the CD4+ cells (3H-thymidine incorporation and cell counts) proved APC properties of MSC, at efficiency comparable to macrophages. Overall, the studies show that the window between APC function and the period at which MSC could become immune suppressive is critical, since activated T-cells could destroy the endothelial barrier between BM and lymphatics/peripheral circulation. These studies show that MSC could be key cells in regulating immune responses in BM, and thereby protect BM from failure.
IFNg-Mediated MHC-II Expression in Mesenchymal Stem Cells (MSC) Is Concentration-Dependent.
Authors: Tang Katherine C. , Kotenko Sergei , Harrison Jonathan S. , Rameshwar Pranela
Published: April 2020

A significant concern in tissue repair is allo-rejection in a host with MHC-II mismatch. Since MSC appear to have veto properties to offset its own rejection, these stem cells may serve as ideal candidates in repair medicine. Interferon-g (IFN-g), which exacerbates immune responses, is known to enhance the expression of MHC-II in antigen presenting cells (e.g., macrophages). This led to the question on the mechanism by which MSC act as immune suppressor cells. We hypothesize that endogenous IFN-γ maintains basal MHC-II expression, while increased levels lead to its down regulation. Indeed, we observed a biphasic response of IFN-γ on the expression of MHC-II. While unstimulated MSC produced 19±5 pg/ml IFN-γ, exogenous IFN-γ (>100 U/ml) led to decreased expression of MHC-II. Suppression of IFN-g by siRNA resulted in undetectable MHC-II. The response of exogenous IFN-γ could not be explained by altered expression of IFN-g receptor type I (IFN-γRI) or changes in STAT-1 activation. However, western blots showed multiple isotypes of the MHC-II master regulator transcription factor, CIITA, in IFN-γ-stimulated MSC, with predominance of CIITA exhibiting low efficiency binding to DNA. This observation might explain why MHC-II is undetectable in the presence of high levels of IFN-γ. These findings support a potential for MSC as an adult stem cell in tissue repair.
Bone Marrow Stroma-Derived Mesenchymal Cell Cultures from Nude/Nude Rat Showing Both Ex Vivo Differentiation Properties and High Metastatic Potential in Nude Mice.
Authors: Wang Mengyu , Kvalheim Gunnar , Mikalsen Svein-Ole , Nesland Jahn M. , Mælandsmo Gunhild , Fodstad Øystein
Published: April 2020

Recent studies have revealed that stem cell like cancer cells can be isolated from different types of primary tumor tissue from brain, breast and skin. Such cells have high capability to form tumors when transplanted into experimental animals. Here we describe a newly developed nude rat mesenchymal stem cellS (rMSCs), which spontaneously changed in vivo properties during culturing. Rat bone marrow cells were isolated from the femur and tibia of a male nude/nude rat and cultured in DMEM medium supplemented with 20% bovine fetal serum (FCS). Non- adherent cells were removed after 72 hours of incubation and the adherent bone marrow cells were cultured until a confluent layer appeared. Adherent cells were detached by trypsin, washed and continued to be cultured. After 23 passages by high-density culture the rMSCs spontaneously became more homogenous and formed spheroids in three-dimensional culture. Following ex vivo culturing under different growth conditions these cells were still able to differentiate into fat and neural like cells. The rMSCs also contain high numbers of colony-forming-unit-fibroblasts(cfu-f). The “tumourigenic” growth pattern observed ex vivo prompted us to examine the rMSCs in vivo. Following subcutaneous injection of 1 x106 cells into nude/nude mice a solid tumor appeared under the skin after 12 days. Moreover, intravenous injections and direct injections into left heart ventricle of rMSCs created solid tumors both in the lung, abdominal cavity, bone and skin. Both phenotypic and genetic characterization of early and late passages of the rMSCs and the tumors formed in the mice are being investigated and more details will be presented at the meeting.
Co-Activation of HOXB7 and BCL2/MYC Via Biallelic IGH Rearrangements in a B-Cell Lymphoma Cell Line.
Authors: Nagel Stefan , Kaufmann Maren , Drexler Hans G. , MacLeod Roderick A.F.
Published: April 2020

Homeobox (Hox) genes mediate tissue-specific expression during development where dysregulation may provoke neoplasia and the involvement of specific Hox genes in hematopoietic malignancies is a recurring theme. In B-cell neoplasias dysregulation may be effected by juxtaposition of Hox genes with immunoglobulin gene regulatory elements, notably IGH. We describe the molecular cytogenetic analysis of a cell line (Epstein-Barr virus negative) established from ascites fluid taken from a 67-year-old male patient at diagnosis of B-cell non-Hodgkin’s lymphoma (follicular, small cleaved cell type). The cell line carries a complex t(8;14;18)(q24;q32;q21) leading to activation of both MYC (at 8q24) and BCL2 (at 18q21) via juxtaposition with different enhancer regions of the IGH locus at 14q32. Both translocated alleles of MYC and BCL2 showed additional genomic amplification in conjunction with IGH by formation of an homogeneously staining region on the der(8). Interestingly, the IGH allele on the second chromosome 14 homolog had also undergone chromosomal juxtaposition, this time with a breakpoint at the HOXB cluster at 17q21 leading to transcriptional activation of HOXB7 but of no other HOXB gene. Although its deregulated expression has been described in certain solid tumor subtypes, notably malignant melanoma, this is the first report describing the involvement of HOXB7 in hematopoietic malignancy. Moreover, the cell line carries a novel internal duplication of chromosome 3, dup(3)(q21q27), effecting fusion of BCL6 with MBNL1, a triplet-expansion repeat gene preferentially expressed during hematopoiesis. Our data thus identify a cell line resource modelling novel hematopoietic rearrangements, notably HOXB7 - an attractive candidate target of recurrent 17q2 rearrangements in B-cell lymphoma.
Decrease of Malignant Potential of t(8;21) Positive Cells after Stable Expression of RUNX-CBFA2T1-Specific Small Interfering RNA.
Authors: Drescher Bettina , Goerlich Kerstin , Reile Dagmar , Heil Gerhard , Heidenreich Olaf , Wagner Katharina , Ganser Arnold , Krauter Juergen
Published: April 2020

The reciprocal chromosomal translocation t(8;21) results in the fusion of the DNA-binding domain of the transcription factor RUNX1 (also called AML1 or CBFα) to CBFA2T1 (also called MTG8 or ETO). The chimeric protein RUNX1-CBFA2T1 interferes with hematopoietic gene expression by recruiting histone deacetylases via N-CoR and mSin3. In addition, impairment of the function of transcription factors such as C/EBPα has been described. To further investigate the function of RUNX1-CBFA2T1 in the development of leukemia, the expression of the fusion protein was inhibited by small interfering RNAs (siRNAs). For stable expression of the siRNA in the target cells, a RUNX1-CBFA2T1-specific siRNA-sequence was cloned into a lentivial vector as short-hairpin-RNA (shRNA). Subsequently, the t(8;21) positive Kasumi-1 cell line was infected with an effeciency of >95% as detected by expression of the GFP reporter gene. As a control, an shRNA targeting luciferase mRNA was used. Expression of the anti-RUNX1-CBFA2T1-shRNA in the Kasumi-1 cells led to a marked reduction of mRNA and protein expression of the fusion gene, whereas the expression of the wildtype RUNX1 gene was not affected. The surface expression of the M-CSF-Receptor, which is a known target gene of C/EBPα, was increased in the RUNX1-CBF2T1 depleted cells. Also, Kasumi-1 cells treated with the shRNA displayed a decrease in CD34 surface expression. In parallel, CD34 mRNA expression was reduced to 10%. To analyze, if CD34 downregulation of the Kasumi-1 cells after RUNX1-CBF2T1 depletion correlates with a loss of progenitor status, the clonogenicity of the cells in semisolid medium was investigated. In Kasumi-1 cells treated with the shRNA against RUNX1-CBFA2T1 the number of spontaneous colonies after 14 days of culture was reduced to about 30% in comparison to cells expressing the control shRNA. In conclusion, these experiments show that RUNX1-CBF2T1 expression can be stably suppressed in t(8;21) positive cells by endogenously expressed shRNAs thereby reducing their clonogenic potential.