Surface antigen expression on CD34+ cord blood cells: Comparative analysis by flow cytometry and limiting dilution (LD) RT-PCR of chymopapain-treated or untreated cells

Biosimilars of granulocyte colony-stimulating factor (G-CSF) have been routinely introduced into clinical practice. However, not functional genomics characterization has been performed yet in comparison with the innovator G-CSF. This study aimed to evaluate the transcriptomic changes in an in vitro model of umbilical cord blood cells (UBC) exposed to G-CSF for the identification of their modulated pathways. Umbilical cord blood cells–derived mononuclear cells (MNCs) were treated with biosimilar and innovator G-CSF for further gene expression profiling analysis using a microarray-based platform. Comparative analysis of biosimilar and innovator G-CSF gene expression signatures allowed us to identify the most commonly modulated pathways by both drugs. In brief, we observed predominantly upmodulation of transcripts related to PI3K-Akt, NF-kappaB, and tumor necrosis factor (TNF) signaling pathways as well as transcripts related to negative regulation of apoptotic process among others. In addition, hematopoietic colony-forming cell assays corroborate the G-CSF phenotypic effects over UBC-derived MNCs. In conclusion, our study suggests that G-CSF impacts UBC-derived cells through the modulation of several signaling pathways associated with cell survival, migration, and proliferation. The concordance observed between biosimilar and innovator G-CSF emphasizes their similarity in regards to their specificity and biological responses.

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Dissecting Macrophage-Derived Microvesicles' Content and Function.

Blood ◽

10.1182/blood.v116.21.3785.3785 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3785-3785

Author(s):

Noura Ismail ◽

Kara Batte ◽

Leni Moldovan ◽

Clay Marsh ◽

Melissa Piper

Keyword(s):

Flow Cytometry ◽

Surface Antigen ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Antigen Expression ◽

Mononuclear Phagocytes ◽

Gm Csf ◽

Wide Range ◽

Surface Antigen Expression ◽

And Function

Abstract Abstract 3785 Microvesicles (MVs) are small membrane-bound vesicles released under normal homeostatic and stimulatory conditions by a wide variety of cell types. Microvesicles are collectively referred to as exosomes and microparticles which vary in size due to different cellular mechanisms responsible for their production. These microvesicles have a wide range of functions from facilitating communication to regulating cellular growth and differentiation. During their production; microvesicles become enriched in various molecules including proteins and nucleic acids. Previously, we have shown that plasma microvesicles derived from many cell lineages contain microRNAs (miRNAs). We also found that the majority of the peripheral blood microvesicles are derived from platelets while those originating from monocytic cells including macrophages represent the second largest population. Since microvesicles derived from mononuclear phagocytes are a large subpopulation in the plasma; we were interested in understanding their content and function. We hypothesized that the content and/or quantity of macrophage-derived microvesicles could induce the maturation of monocytes. To address our hypothesis, peripheral blood monocytes were treated in vitro for 4hr with GM-CSF; washed and cultured in media devoid of cytokines for 24 h then microvesicles were collected. Flow cytometry and electron)confocal microscropy were used to quantify and visualize microvesicles production. To examine the function of the microvesicles on macrophage maturation, the purified microvesicles were then cultured with freshly isolated monocytes. Macrophage differentiation was determined by cellular adherence using a crystal violet uptake assay and changes in surface antigen expression by flow cytometry. We also examined the genetic changes induced in monocytes incubated with the microvesicles compared to GM-CSF-treated cells. We found that freshly isolated monocytes treated with microvesicles from macrophages acquired phenotypic characteristics of a macrophage such as cellular adherence and surface antigen expression. We also found that treatment of naïve monocytes with the microvesicles induced molecular changes similar to GM-CSF treated monocytes. We found more than 7985 mRNAs that were similarly expressed between the two culture conditions. Notably, we observed the unique expression of 1324 and 1079 genes in the GM-CSF-treated compared to the microvesicle-treated cells, respectively. To begin dissecting the molecules contained in the microvesicles responsible for these changes, we performed mass-spectrometry and miRNA profiling. We observed the expression of miRs-223, -222,-191, -484, -016, -026a, and -155 in GM-CSF-derived microvesicles. Notably, these miRNAs were also expressed in the cells from which the microvesicles were released. We have begun bioinformatics analyses to predict whether the expression of the miRNAs may account for the decrease expression of specific genes in cells treated with the microvesicles that undergo differentiation. Many of the proteins found in the vesicles are important in facilitating protein:protein interactions and nucleic acid binding. Based on our observations; we postulate that microvesicles in areas of inflammation may contribute to the inflammatory response through the maturation of immune cells and activation of cells responsible for tissue repair. Thus, defining key components of this response may identify targets to regulate inflammation. Disclosures: No relevant conflicts of interest to declare.

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Restoration of Rituximab Sensitivity in CD20 Negative B-Cell Lymphoma Cells; Epigenetic Change of CD20 Expression after Treatment of Rituximab.

Blood ◽

10.1182/blood.v108.11.4307.4307 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4307-4307

Author(s):

Akihiro Tomita ◽

Junji Hiraga ◽

Hitoshi Kiyoi ◽

Tomohiro Kinoshita ◽

Tomoki Naoe

Keyword(s):

B Cell ◽

Surface Antigen ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Antigen Expression ◽

Molecular Targeting ◽

Rt Pcr ◽

Lymphoma Cells ◽

Cd20 Expression ◽

Surface Antigen Expression

Abstract CD20 surface antigen is widely expressed on normal B-lineage cells and B-lymphoid malignant cells. Rituximab, which is a chimeric monoclonal antibody specifically recognizing CD20 surface antigen, works as a molecular targeting drug against CD20 positive lymphoid malignancies. Although combination chemotherapy with Rituximab has significantly improved the survival of CD20 positive lymphoma patients, CD20 negative tumor regression and transformation becomes a considerable problem. Recently, we have identified a patient with CD20(−) diffuse large B-cell lymphoma (DLBCL) transformed from CD20(+) follicular lymphoma after chemotherapies including Rituximab. Here we established RRBL1 (Rituximab resistant B-cell lymphoma 1) cells from this patient’s CD20(−) B-cell lymphoma cells, and analyzed the mechanisms of negative CD20 surface antigen expression. CD20 was not expressed as determined by immunoblotting and FACS analysis, and resistance to Rituximab was observed after cell culture analysis with/without Rituximab. Expression of wild type CD20 mRNA was confirmed by RT-PCR, and no genetic mutation in coding sequence or promoter region was observed. Quantitative RT-PCR showed that CD20 mRNA expression level was almost 100 times lower than that of CD20 positive B-cell lymphoma cells obtained from DLBCL patients. These data suggest that lower expression level of CD20 mRNA is closely related to the negative CD20 surface antigen expression, and the aberrant transcription regulation by epigenetic mechanisms can be anticipated. With these data, we thought that CD20 expression could be restored by modulating the transcription regulation. As we expected, CD20 protein re-expression was confirmed by enhancing CD20 mRNA expression after the treatment with specific molecular targeting drugs, and the sensitivity for the Rituximab was significantly restored. Chromatin immunoprecipitation assay suggested that recruitment of co-repressor complexes to CD20 promoter might be related to the regulation of CD20 expression. Our data may provide us with a new therapeutic strategy, epigenetic therapy or differentiation therapy, combined with molecular targeting therapy using Rituximab, for Rituximab resistant CD20(−) B-cell malignancies.

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Long Term Culture-Initiating Cells (LTC-IC) Produced from CD34+ Cord Blood Cells with limiting dilution method

Turkish Journal of Hematology ◽

10.5152/tjh.2010.41 ◽

2010 ◽

Author(s):

Gulderen Yanikkaya Demirel ◽

Tulin Budak Alpdogan ◽

Sema Aktas ◽

Mahmut Bayik

Keyword(s):

Cord Blood ◽

Blood Cells ◽

Dilution Method ◽

Long Term Culture ◽

Cord Blood Cells ◽

Limiting Dilution

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Long-term culture-initiating cells (LTC-IC) produced from CD34+ cord blood cells with limiting dilution method

Turkish Journal of Hematology ◽

10.5152/tjh.2010.44 ◽

2010 ◽

Vol 27 (4) ◽

pp. 234-241 ◽

Cited By ~ 1

Author(s):

Gulderen Yanikkaya Demirel ◽

Tulin Budak Alpdogan ◽

Sema Aktas ◽

Mahmut Bayik

Keyword(s):

Cord Blood ◽

Blood Cells ◽

Dilution Method ◽

Long Term Culture ◽

Cord Blood Cells ◽

Limiting Dilution

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Identification of CD34+ Cord Blood Cells and Their Subpopulations in Preterm and Term Neonates Using Three-Color Flow Cytometry

Neonatology ◽

10.1159/000244117 ◽

1994 ◽

Vol 66 (5) ◽

pp. 272-279 ◽

Cited By ~ 12

Author(s):

Bernhard Meister ◽

Martin Tötsch ◽

Andrea Mayr ◽

Martin Widschwendter ◽

Otto Huter ◽

...

Keyword(s):

Flow Cytometry ◽

Cord Blood ◽

Blood Cells ◽

Color Flow ◽

Term Neonates ◽

Cord Blood Cells

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A Quantitative Exploration of Surface Antigen Expression in Common B-Cell Malignancies Using Flow Cytometry

Immunological Investigations ◽

10.1080/08820130500496878 ◽

2006 ◽

Vol 35 (1) ◽

pp. 93-114 ◽

Cited By ~ 63

Author(s):

Scott H. Olejniczak ◽

Carleton C. Stewart ◽

Kathleen Donohue ◽

Myron S. Czuczman

Keyword(s):

Flow Cytometry ◽

B Cell ◽

Surface Antigen ◽

Antigen Expression ◽

B Cell Malignancies ◽

Surface Antigen Expression

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Cord Blood as a Very Valuable Source of Neonatal Cells but Embryonic-Like Nature Reevaluated

Blood ◽

10.1182/blood.v112.11.2897.2897 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2897-2897 ◽

Cited By ~ 2

Author(s):

Anja Buchheiser ◽

Stefanie Liedtke ◽

Amelie Pia Houben ◽

Simon Waclawczyk ◽

Milaid Stephan ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Cord Blood ◽

Blood Cells ◽

Stem Cell Markers ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Cord Blood Cells

Abstract Human umbilical cord blood has become a very valuable source for hematopoietic transplantation. Our group was able to show that CB contains non-hematopoietic stem cells, which were called unrestricted somatic stem cells (USSCs) with a multipotent differentiation potential. These cells have the potential to differentiate into different germ layers (Kögler et al. 2004, Kögler et al. 2005, Kögler et al. 2006, Sensken et al. 2007, Greschat et al. 2008, Ghodsizad A et al. 2008, Trapp et al. 2008). Some studies have now reported a presumably embryonic like nature of cord blood cells. However, Nanog and Oct 4 harbours potential pitfalls for data misinterpretation due to pseudogenes and alternative spliced variants (Liedtke et al. 2007, Liedtke et al. 2008). The related data based on the stem cell markers Nanog and Oct4 concerning these results remain questionable. Therefore, we evaluated the embryonic-like nature of MNCs (n=7), USSC (n=7), CD34+ cells (n=7) derived from cord blood, MNCs from peripheral blood (n=3), MNCs (n=7) and MSCs (n=3) from bone marrow. Using RT-PCR, quantitative RT-PCR and immunohistochemistry, we studied the expression of the pluripotency markers Oct4, Nanog, Sox2 as well as the transcription factors Klf4 and cMyc utilized for the induction of pluripotent stem cells from adult human fibroblasts. The expression level of the transcription factors Klf4 and cMyc was nearly equal in all USSC cell lines and BM MSCs. We neither detected expression of Oct4, Nanog and Sox2 in all tested USSC cell lines nor in MNCs and CD34+ cells from cord blood nor in MSCs, MNCs and CD34+ cells from bone marrow. To increase the sensitivity of our method we performed quantitative Oct4 PCRs. This method revealed that USSCs reach the same Oct4 expression level as human dermal fibroblasts. These results are also supported by the inactive status of the telomerase. As a positive control we used the embryonic carcinoma cell line nTERA-2 showing a high expression of Oct4, Nanog and Sox2. In addition we were able to show that the markers SSEA1, SSEA3 and SSEA4 cannot be used as markers of an embryonic-like phenotype. SSEA-1 recognizes the CD15 epitope, SSEA-4 cross-reacts with an adult MSC subpopulation and SSEA3 was always negative applying the correct isotype controls. However, cord blood does not have to contain embryonic like cells, but it contains neonatal cells as USSC expressing Sox17. For hematopoietic stem cells (HSC) it had already been shown that the transcription factor Sox 17 is required to maintain fetal and neonatal HSC and distinguishes their transcriptional regulation from adult HSCs (Kim et al. 2007). Our results indicate that USSCs and cord blood are neonatal cells without expression of typical embryonic stem cell markers. In more than 10.000 unrelated Cord blood transplants performed so far, no tumor formation associated with an Oct4 positive cell/teratoma formation was observed. Therefore the embryonic-like nature of cord blood cells must be reconsidered.

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