ARTICLE Generation of Human Embryonic Stem Cell-Derived Mesoderm and Cardiac Cells Using Size-Specified Aggregates in an Oxygen-Controlled Bioreactor Sylvia Niebruegge, 1 Ce ´line L. Bauwens, 1,2 Raheem Peerani, 1,2 Nimalan Thavandiran, 1,2 Stephane Masse, 3,4 Elias Sevaptisidis, 3 Kumar Nanthakumar, 3,4 Kim Woodhouse, 2,5 Mansoor Husain, 3,6,7 Eugenia Kumacheva, 8 Peter W. Zandstra 1,2,6,7 1 Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada; telephone: 416-978-8888; fax: 416-978-2666; e-mail: peter.zandstra@utoronto.ca 2 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada 3 Division of Cardiology, Toronto General Hospital, Toronto, Ontario, Canada 4 The Toby Hull Cardiac Fibrillation Management Laboratory, Toronto General Hospital, Toronto, Ontario, Canada 5 Faculty of Applied Science, Queen’s University, Toronto, Ontario, Canada 6 Heart & Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada 7 McEwen Centre for Regenerative Medicine, Toronto General Hospital, Toronto, Ontario, Canada 8 Department of Chemistry, University of Toronto, Toronto, Ontario, Canada Received 4 April 2008; revision received 11 July 2008; accepted 14 July 2008 Published online 25 July 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.22065 ABSTRACT: The ability to generate human pluripotent stem cell-derived cell types at sufficiently high numbers and in a reproducible manner is fundamental for clinical and bio- pharmaceutical applications. Current experimental methods for the differentiation of pluripotent cells such as human embryonic stem cells (hESC) rely on the generation of heterogeneous aggregates of cells, also called ‘‘embryoid bodies’’ (EBs), in small scale static culture. These protocols are typically (1) not scalable, (2) result in a wide range of EB sizes and (3) expose cells to fluctuations in physicochemical parameters. With the goal of establishing a robust bioprocess we first screened different scalable suspension systems for their ability to support the growth and differentiation of hESCs. Next homogeneity of initial cell aggregates was improved by employing a micro-printing strategy to gen- erate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully con- trolled bioreactor system and the impact of oxygen con- centration was investigated. Our results demonstrate the beneficial effects of stirred bioreactor culture, aggregate size- control and hypoxia (4% oxygen tension) on both cell growth and cell differentiation towards cardiomyocytes. QRT-PCR data for markers such as Brachyury, LIM domain homeobox gene Isl-1, Troponin T and Myosin Light Chain 2v, as well as immunohistochemistry and functional analysis by response to chronotropic agents, documented the impact of these parameters on cardiac differentiation. This study provides an important foundation towards the robust gen- eration of clinically relevant numbers of hESC derived cells. Biotechnol. Bioeng. 2009;102: 493–507. ß 2008 Wiley Periodicals, Inc. KEYWORDS: human embryonic stem cells; embryoid bodies; bioreactor; hypoxia; micropatterning; cardiomyo- cytes Introduction Embryonic and pluripotent stem cells are capable of self renewal and multilineage differentiation (Evans and Kauf- Correspondence to: P.W. Zandstra Contract grant sponsor: Heart and Stroke Foundation of Ontario Contract grant sponsor: CHIR Additional Supporting Information may be found in the online version of this article. ß 2008 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 102, No. 2, February 1, 2009 493