Articles by Celine L. Bauwens in JoVE
Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells Celine L. Bauwens*1, Derek Toms*2, Mark Ungrin2 1Institute of Biomaterials and Biomedical Engineering, University of Toronto, 2Department of Comparative Biology and Experimental Medicine, University of Calgary Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.
Other articles by Celine L. Bauwens on PubMed
Generation of Human Embryonic Stem Cell-derived Mesoderm and Cardiac Cells Using Size-specified Aggregates in an Oxygen-controlled Bioreactor Biotechnology and Bioengineering. Feb, 2009 | Pubmed ID: 18767184 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 biopharmaceutical 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 generate large numbers of size-specified hESC aggregates. Finally, these technologies were integrated into a fully controlled bioreactor system and the impact of oxygen concentration 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 generation of clinically relevant numbers of hESC derived cells.
Geometric Control of Cardiomyogenic Induction in Human Pluripotent Stem Cells Tissue Engineering. Part A. Aug, 2011 | Pubmed ID: 21417693 Although it has been observed that aggregate size affects cardiac development, an incomplete understanding of the cellular mechanisms underlying human pluripotent stem cell-derived cardiomyogenesis has limited the development of robust defined-condition cardiac cell generation protocols. Our objective was thus to elucidate cellular and molecular mechanisms underlying the endogenous control of human embryonic stem cell (hESC) cardiac tissue development, and to test the hypothesis that hESC aggregate size influences extraembryonic endoderm (ExE) commitment and cardiac inductive properties. hESC aggregates were generated with 100, 1000, or 4000 cells per aggregate using microwells. The frequency of endoderm marker (FoxA2 and GATA6)-expressing cells decreased with increasing aggregate size during early differentiation. Cardiogenesis was maximized in aggregates initiated from 1000 cells, with frequencies of 0.49±0.06 cells exhibiting a cardiac progenitor phenotype (KDR(low)/C-KIT(neg)) on day 5 and 0.24±0.06 expressing cardiac Troponin T on day 16. A direct relationship between ExE and cardiac differentiation efficiency was established by forming aggregates with varying ratios of SOX7 (a transcription factor required for ExE development) overexpressing or knockdown hESCs to unmanipulated hESCs. We demonstrate, in a defined, serum-free cardiac induction system, that robust and efficient cardiac differentiation is a function of endogenous ExE cell concentration, a parameter that can be directly modulated by controlling hESC aggregate size.
Scalable Cardiac Differentiation of Human Pluripotent Stem Cells As Microwell-generated, Size Controlled Three-dimensional Aggregates Methods in Molecular Biology (Clifton, N.J.). 2014 | Pubmed ID: 25070323 The formation of cells into more physiologically relevant three-dimensional multicellular aggregates is an important technique for the differentiation and manipulation of stem cells and their progeny. As industrial and clinical applications for these cells increase, it will be necessary to execute this procedure in a readily scalable format. We present here a method employing microwells to generate large numbers of human pluripotent stem cell aggregates and control their subsequent differentiation towards a cardiac fate.