We recently reported that iPSCs can be generated using non
We recently reported that iPSCs can be generated using non-modified mRNA (Arnold et al., 2012). In this report we will focus on the crucial steps for successfully applying this method.
Material and methods
Acknowledgments The work was supported by funding from the German Federal Ministry of Education and Research (BMBF 1315883). The publication costs were funded by the University of Leipzig. The teratoma assays have been supported by the Deutsche Forschungsgemeinschaft (SFB 1002, TP C05). The authors thank the IZKF core unit DNA technologies of Dr. Krohn at the University of Leipzig for performing gene array experiments.
Introduction Transcriptional regulation is of particular relevance for self-renewal of hematopoietic stem Asiatic acid and accurate hematopoietic differentiation. The potential to give rise to mature hematopoietic cells as well as to preserve stem cell properties is regulated by a set of transcription factors, of which only few are well-characterized (Orkin and Zon, 2008). The ETS transcription factor GA-binding protein (GABP) was recently shown to play a crucial role for myeloid differentiation in mice and humans (Yang et al., 2011; Yu et al., 2011; Ripperger et al., 2015). Moreover, GABP was reported to directly impact propagation of leukemic clones in mice transplanted with BCR-ABL1+ murine leukemic stem cells, which resemble human chronic myeloid leukemia (CML) (Yu et al., 2012; Yang et al., 2013). Our recent work supported these observations by showing that GABP affects viability and imatinib sensitivity in human CML cell lines (Manukjan et al., 2015). Here, we focus on the effects of GABP in human normal and leukemic hematopoietic stem/progenitor cells and demonstrate that GABP is required for proper myeloid differentiation in human primary cells. GABP functions as an obligate heterodimer, in which the alpha-subunit (GABPα) binds targeted DNA-motifs and the beta-subunit (GABPβ1) contributes to transcriptional regulation via its transactivation capacity (Rosmarin et al., 2004). Hence, ectopic overexpression of GABPβ1 lacking the transcriptional activation domain (TAD) results in a dominant-negative GABP, as we show here and have already demonstrated in the preliminary work (Manukjan et al., 2015). In this context, we further show that impaired GABP decreases leukemic stem/progenitor cell capacity of human CD34+ cells derived from CML patients.
Material & methods Mobilized CD34+ cells were purified from peripheral blood leukapheresis material from three individuals in hematological remission after treatment for acute myeloid leukemia (AML). Bone marrow CD34+ cells were obtained from healthy donors. Bone marrow CD34+ cells from five CML patients (three females; two males; age range at diagnosis: 44–87years) in chronic phase were obtained at the time of diagnosis. Patients did not receive tyrosine kinase inhibitor therapy prior to sampling. Diagnosis of CML was confirmed by standard cytogenetic and molecular genetic analyses to detect translocation t(9;22)(q34;q11) and detection and quantification of p210 BCR-ABL1 fusion transcripts as described previously (Emig et al., 1999; Hughes et al., 2006). The investigation was approved by the Hannover Medical School Ethics Committee (No. 2899) and written consent was obtained from each patient in accordance with the Declaration of Helsinki. In all cases, CD34+ cells were purified by magnetic separation according to the manufacturer\'s recommendations (Miltenyi Biotec, Bergisch Gladbach, Germany). Cultivation, transduction and chemical treatment (DMSO and imatinib) of K-562 cells (ACC-10, DSMZ, Braunschweig, Germany) were performed as described earlier (Manukjan et al., 2015). The pRSF91.IRES.dTomato.pre* vector was used to ectopically overexpress GABPB1.ΔTAD, following published protocols (Ripperger et al., 2015). CD34+ cells were transduced with VSV-G pseudotyped retroviral particles using a RetroNectin protocol (Takara Bio Europe/Clontech, Saint-Germain-en-Laye, France) and a multiplicity of infection (MOI) of 50. Quantitative RT-PCR was performed using the QuantiTect SYBR Green RT-PCR Master Mix (Qiagen, Hilden, Germany) under standard conditions. Relative expression was calculated by the ΔΔCt method in correlation to SDHA. Primer sequences are as follows: GABPB1 (forward: 5′-GGT CAA GAT GAT GAA GTT CG-3′; reverse: 5′-CTG GCA TCT CTG CTC ACA C-3′); SDHA (forward: 5′-GCC ATC CAC TAC ATG ACG-3′; reverse: 5′-TCC ATA TAA GGT GTG CAA TAG C-3′).