br Materials and methods br Acknowledgments This study was s

Materials and methods
Acknowledgments This study was supported by postdoctoral research station of Central South University and funded by China Postdoctoral Science Foundation (2014M560650 to S.Z.), the National Natural Science Foundation of China (grant number 31401248 to S.Z.). We thank the genetic laboratory of the Reproductive & Genetic Hospital of CITIC-Xiangya for their assistance.
Resource table. Resource details Chronic myeloid leukemia (CML) is a malignant myeloproliferative disorder originating from a primitive hematopoietic stem cell leading to an expansion of the progenitors and differentiated myeloid purchase ABT-888 in the peripheral blood (Chomel and Turhan, 2011). Despite the major progress obtained in this disease by the use of tyrosine kinase inhibitors, the most primitive stem cells are resistant to these therapies (Jiang et al., 2007; Chomel et al., 2011). We have generated iPSC from the cryopreserved diagnostic sample of a patient according to his clinical response to Imatinib, the first clinically licensed tyrosine kinase inhibitor. This 14-year old young patient exhibited a clear resistance to this drug with absence of molecular response after 18months of starting therapy. There was no mutation in the ABL-kinase domain of the BCR-ABL gene. In 2005, given his young age and the availability of a HLA-compatible sibling, an allogeneic stem cell transplant was performed with success. In 2016, he remains in good health in complete haematological and molecular remission. An induced pluripotent stem cell (iPSC) line were generated using cryopreserved CD34+ leukemic cells collected at diagnosis by non-integrative transduction of Oct3/4, Sox2, Klf4, and cMyc (Takahashi et al., 2007). iPSC colonies growing on MEF stromal layers were morphologically identified after 2–4weeks (Fig. 1A), individually plucked as well as a polyclonal stock and expanded. Besides the polyclonal PB32 cell line, 3 clones (PB32–1, PB32–4 and PB32–5) were expanded. All have been characterized for their pluripotency. They exhibited typical markers of pluripotency such as expression of SSEA4 and TRA-1-60 (Fig. 1B). Cytogenetics analysis demonstrated the presence of Ph1 chromosome (Fig. 1C). Teratoma assays showed the presence of normal differentiation towards endodermal, ectodermal and mesodermal layers (Fig. 1D).
Materials and methods
Acknowledgements
Resource table. Resource details Large amounts of highly pure and functionally intact cardiomyocytes (CMs) are required for applications in tissue engineering, drug discovery and toxicity testing. The most efficient system for production of pure CMs at large quantities is in suspension mass cultures (Hemmi et al., 2014; Kempf et al., 2014; Nguyen et al., 2014; Schroeder et al., 2005) and relies on regulated expression of exogenous reporter and/or selection markers under the control of a cardiospecific promoter (Anderson et al., 2007; Friedrichs et al., 2015; Kita-Matsuo et al., 2009; Klug et al., 1996; Kolossov et al., 2006; Ritner et al., 2011; van Laake et al., 2010). Here, we have genetically modified murine induced pluripotent stem cell (miPSC) line TiB7.4 (Meissner et al., 2007) to obtain the transgenic line αPIG-AT25 by transfecting the cells with the plasmid expressing puromycin N-acetyltransferase (PAC) and enhanced green fluorescent protein (EGFP) under the control of the CM-specific α-myosin heavy chain (α-MHC) promoter (Fig. 1A). The αPIG-AT25 miPSCs exhibit pluripotent stem cell colony morphology (Fig. 1B) and express alkaline phosphatase (Fig. 1C), the self-renewal protein Oct4 (Fig. 1D) and pluripotent stem cell marker SSEA1 (Fig. 1E). The promoter regions of Nanog and Oct4 genes are similarly hypomethylated in αPIG-AT25 iPSCs and transgenic murine embryonic stem cells (ESC) line αPIG44 that was generated previously using the same vector (Kolossov et al., 2006) (Fig. 1F). In contrast, the same promoter regions were highly methylated in tail tip fibroblasts (TTF), from which the iPSCs were generated, and in αPIG-AT25 miPSC-derived CMs (Fig. 1F).