293 MASS PRODUCTION OF Nkx2.5-POSITIVE CARDIAC PROGENITOR CELLS DERIVED FROM MOUSE EMBRYONIC STEM CELLS IN SLOW-TURNING LATERAL VESSEL FOR CELL TRANSPLANTATION AND DRUG TESTING

Abstract

Regenerative cell therapy against cardiovascular disease would require mass production and purification of specific cell types before transplantation. To enable large-scale production of embryonic stem (ES)-derived pure cardiomyocytes, we developed an animal model for a single-step scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled slow-turning lateral vessel (STLV, Synthecon, Inc., Houston, TX, USA) bioreactor following inoculation with a single cell suspension of mouse ES cells. To enhance the yield of cardiac progenitor cells, mouse ES cells (HM1; 129Sv/Ola, Magin et al. 1992 Nucl. Acids Res. 20, 3795–3796) were targeted with the cardiac-specific mouse Nkx2.5 promoter driven enhanced fluorescent green protein (EGFP). Among 15 targeted colonies, which were characterised based on morphology, the ability to form EB, EGFP expression, and in vitro differentiation ability toward cardiomyocytes, 3 lines were further evaluated for the efficiency of cardiomyocyte production. The 3 lines were cultured in STLV bioreactor and compared with classical hanging drop (HD) and static suspension culture methods. Embryonic bodies at day 3 to 8 were collected and analysed by using fluorescence-activated cell sorting for markers of pluripotency (e.g. Oct-4, SSEA1, Nanog) and cardiac (e.g. Nkx2.5, Troponin T) lineage commitments. Data was analysed by one-way ANOVA and t-tests. The results showed that both level and kinetics of Nkx2.5 expression was dependent on culture conditions. The STLV and static suspension culture methods produced higher rates of Nkx2.5-positive cells on day 5 than that of HD (70 and 54 v. 30%, respectively). The STLV method produced a highly uniform population of efficiently differentiating EB in large quantities and resulted in the highest, 108 yield of cardiomyocytes in a single 110-mL STLV on day 4. In conclusion, the STLV method provides a technological platform for controlled large-scale generation of ES-cell-derived cardiomyocytes for clinical and industrial applications. In vivo transplantation tests of cardiomyocytes produced via STLV are currently underway. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205 and NKTH/KPI (NKFP_07_1-ES2HEART-HU OM-00202-2007), CHE-TRF senior scholarship, No. RTA 5080010 (M.T.), and the Thailand Commission on Higher Education [CHE-PhD-SW-2005-100 (S.R.), CHE-PhD-SW-RG-2007 (N.K.)].