Abstract 5436: Generation of Induced Pluripotent Stem Cells From Peripheral Blood of Coronary Artery Disease Patients
Background. Studies have demonstrated that ectopic expression of defined transcription factors can reprogram somatic cells into induced pluripotent stem cells (iPS). However, the sources of patient-derived iPS cells are limited to fibroblasts or mesenchymal cells, which require invasive procedures and long-term manipulation. Accordingly, in this study we aimed to generate human iPS cells from peripheral blood mononuclear cells (PB-MNCs) of coronary artery disease (CAD) patients.
Methods and Results. We recruited one healthy volunteer and three male patients aged 58, 62, and 77, respectively, who have CADs confirmed by coronary angiography. PB-MNCs obtained by density gradient centrifugation were cultured in an adherent fashion. The adherent cells were infected with the lentiviral supernatant for the four transcription factors of OCT4, SOX2, KLF4 or c-MYC. We used third generation lentiviral vectors which do not have infectivity. At 10 days post-infection, colonies showing hES cell-like morphology were selected and transferred to feeder layers for expansion. After 3 to 4 passages, we observed tightly packed and flat colonies of cells characterized by large nuclei and scant cytoplasm, highly suggesting successful generation of iPS cells from PB-MNCs of all the patients (HiPS-ECP1, HiPS-ECP2 and HiPS-ECP3) and a healthy volunteer (HiPS-ENB). A series of assays including cytochemistry, real-time RT-PCR and teratoma formation assay revealed identical pluripotency of these iPS cells to human ES cells (H1). Bisulfite sequencing of the OCT4 and NANOG promoters in the iPS cells showed profound demethylation similar to hES cells. We next examined the differentiation capacity of the iPS cells. The iPS cells readily form EBs at 10 days in suspension culture. Upon further cultivation of EBs for another 10 days, these iPS cells spontaneously differentiated into cell types of all 3 embryonic germ layers.
In conclusion. This study provides the first evidence that iPS cells can be successfully generated from PB of CAD patients, which have almost identical characteristics to iPS cells derived from healthy volunteers or human ES cells. This advance will facilitate the use of iPS cells for autologous cell therapy for ischemic cardiovascular diseases.