Generation of highly purified human cardiomyocytes from peripheral blood mononuclear cell-derived induced pluripotent stem cells.

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

Bronchoepithelial expression of CXCR1 and CXCR2 does not facilitate transepithelial migration of neutrophils.

BACKGROUND: Neutrophilic airway inflammation is one of the key features of chronic obstructive pulmonary disease (COPD). The chemokine receptors 1 (CXCR1) and 2 (CXCR2) are expressed in the bronchial mucosa during chronic inflammation and might be of importance for transepithelial migration of neutrophils. OBJECTIVES: This study addressed the role of bronchoepithelial CXCR1 and CXCR2 expression with respect to transepithelial migration of neutrophils. METHODS: Primary bronchial epithelial cells (PBECs) derived from COPD patients and healthy controls as well as transiently CXCR1- and CXCR2-transfected Calu-6 cells were used for transepithelial migration assays of neutrophils under various conditions. Epithelial CXCR1 and CXCR2 expression was verified by means of flow cytometry. RESULTS: Transepithelial migration of neutrophils was significantly increased following lipopolysaccharide pretreatment of epithelial cells. Transient transfection of CXCR1 and CXCR2 neither augmented the transepithelial migration of neutrophils, nor did the selective blockade of CXCR1 and CXCR2 have any significant effect on neutrophilic transepithelial migration. In addition, no differences were found in PBECs and neutrophils derived from healthy controls and COPD patients. CONCLUSIONS: The data of the present study do not support the hypothesis that bronchoepithelial expression of CXCR1 and/or CXCR2 facilitate transepithelial migration of neutrophils.

Expression of CXC chemokine receptors 1 and 2 in human bronchial epithelial cells.

INTRODUCTION: CXC chemokine receptor 1 (CXCR1) and CXC chemokine receptor 2 (CXCR2) have been shown to play an important role in transepithelial migration of neutrophil granulocytes during inflammation in various tissues. This study investigated the regulation of gene expression and surface expression of CXCR1 and CXCR2 in a human bronchial epithelial cell line (BEAS-2B), as well as in primary bronchial epithelial cells (PBECs) from 10 COPD patients and 10 control subjects. METHODS AND RESULTS: The transcription expression of CXCR1 and CXCR2 was quantitatively assessed by means of real-time polymerase chain reaction (PCR) under various inflammatory conditions. Flow cytometry was used to measure CXCR1 and CXCR2 surface expression. There was a low baseline expression of CXCR1 and CXCR2 in real-time PCR in PBECs from COPD patients and control subjects as well as in BEAS-2B cells, and no significant regulation occurred under various inflammatory conditions in PBECs and BEAS-2B cells. Furthermore, unstimulated surface expression of CXCR1 and CXCR2 on BEAS-2B cells was very low, and no significant regulation was detectable under time-dependent inflammatory stimulation up to 24 h. CONCLUSION: Various inflammatory responses that are of potential relevance in COPD pathophysiology do not affect transcription regulation and surface expression of the interleukin-8 receptors CXCR1 and CXCR2 on human bronchial epithelial cells.