Highlights

We present a strategy to optimize cardiac differentiation in suspension for hiPSCs.

The matrix-free suspension platform integrates hPSC expansion and differentiation.

Cardiac production in suspension achieves >90% purity with 1L spinner flasks.

The production process in suspension is defined, scalable, and GMP compliant.

To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2109 CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

Myocardial infarction and heart failure are leading causes of death worldwide. As the myocardium has a very limited regenerative capacity, endogenous cell regeneration cannot adequately compensate for heart damage caused by myocardial infarction. The concept of cell replacement therapy is an appealing approach to the treatment of these cardiac diseases. HPSCs are an attractive cell source for cell replacement therapies because they can be expanded indefinitely in culture and efficiently differentiated into a variety of cell lineages, including cardiac cells. However, current hPSC expansion and differentiation methods rely on adherent cell culture systems that are challenging to scale up to the levels required to support pre-clinical and clinical studies.

Activin/Nodal/TGF-, BMP, and Wnt signaling play pivotal roles in regulating mesoderm and cardiac specification during embryo development (Arnold and Robertson, 2009, Buckingham et al., 2005, Tam and Loebel, 2007, David et al., 2008, Naito et al., 2006, Ueno et al., 2007andBurridge et al., 2012). Significant progress has been made in the cardiac differentiation process by modulating Activin, BMP, and Wnt pathways, which can efficiently drive differentiation to over 80% purity of CM (Burridge et al., 2014, Kattman et al., 2011, Lian et al., 2012, Yang et al., 2008, Zhang et al., 2012andZhu et al., 2011). Using an adherent cell culture platform, one study revealed that using 2 small Wnt pathway modulators to sequentially activate and then inhibit Wnt signaling at different differentiation stages of the culture is sufficient to drive cardiac differentiation and generate CM with high purity (Lian et al., 2012). In spite of this, adherent culture systems have limited scalability and are not practical to support the anticipated CM requirements of clinical trials. Alternatively, using an embryoid body (EB) differentiation method, a complex cardiac induction procedure involving stage-specific treatments with growth factors and small molecules to modulate Activin/Nodal, BMP, and Wnt pathways has been reported to be effective in cardiac differentiation in a suspension culture system (Kattman et al., 2011andYang et al., 2008). However, the process of generating EBs is inefficient, rendering this method impractical for large scale CM production. An additional limitation of these approaches for scale-up application is that both methods are based on the expansion of the hPSCs in adherent culture and the subsequent CM differentiation process in either adherent culture or as EBs. The labor intensiveness and limited scalability of current processes have been the primary bottle necks to the large scale production of CM for clinical applications of hPSC-derived CM.

Pre-clinical studies suggest that doses of up to one billion CM will be required to achieve therapeutic benefit after transplantation (Chong et al., 2014andLaflamme and Murry, 2005). In order to meet the current CM demand for pre-clinical studies and the anticipated demand for foreseeable clinical studies, development of a robust, scalable and cGMP-compliant differentiation process for the production of both hPSCs and hPSC-derived CM is essential. Suspension cell culture is an attractive platform for large scale manufacture of cell products for its scale-up capacity. Application of a suspension culture platform to support hPSC growth in matrix-free cell aggregates has been well established (Amit et al., 2010, Krawetz et al., 2010, Olmer et al., 2010, Singh et al., 2010, Steiner et al., 2010andChen et al., 2012). We previously also reported the development of a defined, scalable and cGMP-compliant suspension system to culture hPSCs in the form of cell aggregates (Chen et al., 2012). With this suspension culture system, hPSC cultures can be serially passaged and consistently expanded. In the present study we adapted our suspension culture system to establish a robust, scalable and cGMP-compliant process for manufacturing CM. We were able to use hPSC aggregates generated in the suspension culture system directly to produce CM with high efficiency and yield in suspension with various scales of spinner flasks. We optimized various critical process parameters including: small molecule concentration, induction timing and agitation rates for differentiation cultures in spinner flasks with scales up to 1L. In this study, we integrated undifferentiated hPSC expansion and small molecule-induced cardiac differentiation into a scalable suspension culture system using spinner flasks, providing a streamlined and cGMP-compliant process for scale-up CM differentiation and production.

We routinely maintained the hPSCs lines H7 (WA07, WiCell), ESI-017 (BioTime), and a hiPSC line (a gift from Dr. Joseph Wu, Stanford) in the form of cell aggregates in suspension culture as previously described (Chen et al., 2012). Briefly, suspension-adapted hPSCs were seeded as single cells at a density of 2.53105 cells/mL in 125, 500, or 1000mL spinner flasks (Corning) containing culture medium (StemPro hESC SFM, Thermo Fisher Scientific, Life Technologies) with 40ng/mL bFGF (Life Technologies) and 10M Y27632 (EMD Millipore). Stirring rates were adjusted to between 5070rpm depending on the vessel size and hPSC line. Medium was changed every day by demi-depletion with fresh culture medium without Y27632. Cells were dissociated with Accutase (Millipore) into single cells and passaged every 34days when the aggregate size reached approximately 300m. Cell suspension cultures were maintained in 5% CO2 with 95% relative humidity at 37C.

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Development of a scalable suspension culture for cardiac ...

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