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A microfluidic platform for dynamic cell culture and quantitative time-lapse imaging of non-adherent cells
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
3036 - NON-B-CELL FUNCTION OF EBF1 IN THE HEMATOPOIETIC STEM CELL NICHE Marta Derecka and Rudolf Grosschedl Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSCs is still a major challenge because the cues from stromal cells of the bone marrow, including mesenchymal stem and progenitor cells (MSPC) that regulate hematopoietic homeostasis remain largely elusive. Here, we identified early Bcell factor 1 (Ebf1) as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblastic colonies (CFU-F) and impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. Prx1-Cre-mediated deletion of Ebf1 in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. We also observed a reduced ability of HSCs from Prx1-Cre/Ebf1flx/flx mice to form colonies in methylcellulose. Finally, adoptive transfers of wild type HSPCs to Ebf1+/recipients showed a decrease of the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs.
3037 - A MICROFLUIDIC PLATFORM FOR DYNAMIC CELL CULTURE AND QUANTITATIVE TIME-LAPSE IMAGING OF NON-ADHERENT CELLS Philip Dettinger1,2,3, Martin Etzrodt1, Tino Frank1, Nouraiz Ahmed1, Andreas Reimann1, Christoph Trenzinger1, Laura Skylaki1, Oliver Hilsenbeck1, Dirk Loeffler1, Konstantinos Kokkaliaris1, Savas Tay1, and Timm Schroeder1 1 ETHZ, Zurich, Switzerland; 2BSSE, Switzerland; 3CSD, Switzerland Environmental challenges presented to single cells can be interpreted by signalling cascades and influence their long-term fates and functions. Demonstrating the influence of these external stimuli on cellular fate requires the ability to continuously observe single cells under dynamically controlled conditions over long timeframes. However, in the case of non-adherent cells the capacity to conduct such experiments has been lacking because any applied media flow can easily displace such cells and make the maintenance of their identity over time impossible. Here we present an automated microfluidic chip designed for the culture of non-adherent cells in dynamically changing cell culture conditions across several days. This valve controlled design enables targeted seeding of cells in up to 48 independently controlled culture chambers which provide sufficient space for colony expansion. Diffusion based media exchange occurs rapidly and minimizes displacement of cells. Furthermore, the low chamber height minimizes autofluorecence effects of cell culture media enabling improved quantification of fluorescent reporters. Primary hematopoietic stem and progenitor cells and murine embryonic stem cells were used to validate the function of this device. This novel cell culture platform will have important applications to address questions in cell signalling dynamics and cell fate choices related to changes in environmental signalling.
S67
3038 - POLYDIMETHYLSILOXANE (PDMS) MATERIAL COMMONLY USED IN MICROBIOREACTORS ABSORBS RETINOIC ACID AND MODIFIES HAEMATOPOIETIC CELL PHENOTYPE Michael Doran1,2, Kathryn Futrega1, Jianshi Yu3, Jace Jones3, Maureen Kane3, William Lott1, and Kerry Atkinson1 1 Queensland University of Technology at the Translational Research Institute, Woolloongabba, Australia; 2Mater Medical Research - University of Queensland, Australia; 3Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, USA Polydimethylsiloxane (PDMS) is commonly used in the fabrication of microdevices or microbioreactors used to study the biological potential of single haematopoietic cells, or as a material in the fabrication of bioreactors for large-scale haematopoietic cell expansion. When umbilical cord blood (CB)-derived CD34+ cells were expanded in PDMS culture devices, they exhibited reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. In this presentation we describe a series of experiments, which demonstrate that ATRA-mediated CD38 expression is attenuated when cultures are maintained on PDMS. Pre-incubation of medium on PDMS results in a time-dependant depletion of ATRA and increasingly attenuated CD38 expression. Finally, we used Ingenuity Pathway Analysis (IPA) to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34+ haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithe- lial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a) all in standard culture media. Analysis across all of these cell lines indicated that ATRA was the most likely common upstream regulator of perturbed pathways. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced ex- pression of CD38 on the cell surface of CB-derived CD34+ cells. This is an important result, that should be considered by those contemplating using PDMS-based culture devices to study haematopoietic cells.
3039 - REPROGRAMMING OF PRIMARY HUMAN PHILADELPHIA CHROMOSOME-POSITIVE B CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS INTO NONLEUKEMIC MACROPHAGES Christopher Dove, Miles Linde, Scott McClellan, and Ravi Majeti Stanford University, Stanford, USA BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (BCR-ABL1+ B-ALL) is an aggressive hematopoietic neoplasm characterized by a block in differentiation due in part to the somatic loss of transcription factors required for B cell development. We hypothesized that overcoming this differentiation block by forcing cells to reprogram to the myeloid lineage would reduce the leukemogenicity of these cells. We found that primary human BCR-ABL1+ B-ALL cells could be induced to reprogram into macrophage-like cells by exposure to myeloid differentiation-promoting cytokines in vitro or by transient expression of the myeloid transcription factors C/ EBPa or PU.1. The resultant cells were clonally related to the primary leukemic blasts but resembled normal macrophages in appearance, immunophenotype, gene expression, and function. Most importantly, these macrophage-like cells were unable to establish disease in xenograft hosts, indicating that lineage reprogramming eliminates the leukemogenicity of BCR-ABL1+ B-ALL cells and suggesting a previously unidentified therapeutic strategy for this disease. We determined that myeloid reprogramming may occur to some degree in human patients by identifying primary CD14+ monocytes/macrophages in BCR-ABL1+ B-ALL patient samples that possess the BCR-ABL1+ translocation and clonally recombined VDJ regions. Most recently, we have polarized B-ALL blasts to dendritic cell and macrophage subsets for potential immunotherapeutic use. We are also investigating small molecules that may more efficiently reprogram blasts than myeloid cytokines alone.
3036 - NON-B-CELL FUNCTION OF EBF1 IN THE HEMATOPOIETIC STEM CELL NICHE Marta Derecka and Rudolf Grosschedl Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Hematopoietic stem and progenitor cells (HSPC) are in daily demand worldwide because of their ability to replenish entire blood system. However, the in vitro expansion of HSCs is still a major challenge because the cues from stromal cells of the bone marrow, including mesenchymal stem and progenitor cells (MSPC) that regulate hematopoietic homeostasis remain largely elusive. Here, we identified early Bcell factor 1 (Ebf1) as new transcription regulator of MSPCs activity. Mesenchymal progenitors isolated from Ebf1-/- mice show diminished capacity to form fibroblastic colonies (CFU-F) and impaired in vitro differentiation towards osteoblasts, chondrocytes and adipocytes. Prx1-Cre-mediated deletion of Ebf1 in MSPCs of mice leads to reduced frequency and numbers of HSPCs and myeloid cells in the bone marrow. We also observed a reduced ability of HSCs from Prx1-Cre/Ebf1flx/flx mice to form colonies in methylcellulose. Finally, adoptive transfers of wild type HSPCs to Ebf1+/recipients showed a decrease of the absolute numbers of HSPCs in primary recipients and reduced donor chimerism within the HSCP compartment in competitive secondary transplant experiments. Thus, our study establishes Ebf1 as a novel regulator of MSPCs playing a crucial role in the maintenance and differentiation of HSPCs.
3037 - A MICROFLUIDIC PLATFORM FOR DYNAMIC CELL CULTURE AND QUANTITATIVE TIME-LAPSE IMAGING OF NON-ADHERENT CELLS Philip Dettinger1,2,3, Martin Etzrodt1, Tino Frank1, Nouraiz Ahmed1, Andreas Reimann1, Christoph Trenzinger1, Laura Skylaki1, Oliver Hilsenbeck1, Dirk Loeffler1, Konstantinos Kokkaliaris1, Savas Tay1, and Timm Schroeder1 1 ETHZ, Zurich, Switzerland; 2BSSE, Switzerland; 3CSD, Switzerland Environmental challenges presented to single cells can be interpreted by signalling cascades and influence their long-term fates and functions. Demonstrating the influence of these external stimuli on cellular fate requires the ability to continuously observe single cells under dynamically controlled conditions over long timeframes. However, in the case of non-adherent cells the capacity to conduct such experiments has been lacking because any applied media flow can easily displace such cells and make the maintenance of their identity over time impossible. Here we present an automated microfluidic chip designed for the culture of non-adherent cells in dynamically changing cell culture conditions across several days. This valve controlled design enables targeted seeding of cells in up to 48 independently controlled culture chambers which provide sufficient space for colony expansion. Diffusion based media exchange occurs rapidly and minimizes displacement of cells. Furthermore, the low chamber height minimizes autofluorecence effects of cell culture media enabling improved quantification of fluorescent reporters. Primary hematopoietic stem and progenitor cells and murine embryonic stem cells were used to validate the function of this device. This novel cell culture platform will have important applications to address questions in cell signalling dynamics and cell fate choices related to changes in environmental signalling.
S67
3038 - POLYDIMETHYLSILOXANE (PDMS) MATERIAL COMMONLY USED IN MICROBIOREACTORS ABSORBS RETINOIC ACID AND MODIFIES HAEMATOPOIETIC CELL PHENOTYPE Michael Doran1,2, Kathryn Futrega1, Jianshi Yu3, Jace Jones3, Maureen Kane3, William Lott1, and Kerry Atkinson1 1 Queensland University of Technology at the Translational Research Institute, Woolloongabba, Australia; 2Mater Medical Research - University of Queensland, Australia; 3Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, USA Polydimethylsiloxane (PDMS) is commonly used in the fabrication of microdevices or microbioreactors used to study the biological potential of single haematopoietic cells, or as a material in the fabrication of bioreactors for large-scale haematopoietic cell expansion. When umbilical cord blood (CB)-derived CD34+ cells were expanded in PDMS culture devices, they exhibited reduced CD38 surface expression, relative to cells cultured on tissue culture polystyrene (TCP). All-trans retinoic acid (ATRA) induces CD38 expression, and we reasoned that this hydrophobic molecule might be absorbed by PDMS. In this presentation we describe a series of experiments, which demonstrate that ATRA-mediated CD38 expression is attenuated when cultures are maintained on PDMS. Pre-incubation of medium on PDMS results in a time-dependant depletion of ATRA and increasingly attenuated CD38 expression. Finally, we used Ingenuity Pathway Analysis (IPA) to identify potential upstream regulators. This analysis was performed for differentially expressed genes in primary cells including CD34+ haematopoietic progenitor cells, mesenchymal stromal cells (MSC), and keratinocytes, and cell lines including prostate cancer epithe- lial cells (LNCaP), breast cancer epithelial cells (MCF-7), and myeloid leukaemia cells (KG1a) all in standard culture media. Analysis across all of these cell lines indicated that ATRA was the most likely common upstream regulator of perturbed pathways. We demonstrate here that ATRA is absorbed by PDMS in a time-dependent manner and results in the concomitant reduced ex- pression of CD38 on the cell surface of CB-derived CD34+ cells. This is an important result, that should be considered by those contemplating using PDMS-based culture devices to study haematopoietic cells.
3039 - REPROGRAMMING OF PRIMARY HUMAN PHILADELPHIA CHROMOSOME-POSITIVE B CELL ACUTE LYMPHOBLASTIC LEUKEMIA CELLS INTO NONLEUKEMIC MACROPHAGES Christopher Dove, Miles Linde, Scott McClellan, and Ravi Majeti Stanford University, Stanford, USA BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (BCR-ABL1+ B-ALL) is an aggressive hematopoietic neoplasm characterized by a block in differentiation due in part to the somatic loss of transcription factors required for B cell development. We hypothesized that overcoming this differentiation block by forcing cells to reprogram to the myeloid lineage would reduce the leukemogenicity of these cells. We found that primary human BCR-ABL1+ B-ALL cells could be induced to reprogram into macrophage-like cells by exposure to myeloid differentiation-promoting cytokines in vitro or by transient expression of the myeloid transcription factors C/ EBPa or PU.1. The resultant cells were clonally related to the primary leukemic blasts but resembled normal macrophages in appearance, immunophenotype, gene expression, and function. Most importantly, these macrophage-like cells were unable to establish disease in xenograft hosts, indicating that lineage reprogramming eliminates the leukemogenicity of BCR-ABL1+ B-ALL cells and suggesting a previously unidentified therapeutic strategy for this disease. We determined that myeloid reprogramming may occur to some degree in human patients by identifying primary CD14+ monocytes/macrophages in BCR-ABL1+ B-ALL patient samples that possess the BCR-ABL1+ translocation and clonally recombined VDJ regions. Most recently, we have polarized B-ALL blasts to dendritic cell and macrophage subsets for potential immunotherapeutic use. We are also investigating small molecules that may more efficiently reprogram blasts than myeloid cytokines alone.