Human pluripotent stem cells (hPSCs) can be utilized as a source of cardiomyocytes for heart therapies. However, realization of the clinical potential of hPSCs will require efficient and cost-effective bioprocesses for the large-scale generation of implantable cardiomyocytes. We demonstrated the production of cardiac cells from encapsulated embryonic stem cells cultured in stirred-suspension bioreactors1. More recently, we established differentiation protocols for directing the fate of hPSCs along cardiac cell lineages utilizing physiological factors. Importantly, application of these protocols was investigated for the scalable production of heart muscle cells in stirred-suspension microcarrier vessels.
A 4-stage differentiation scheme was established for guiding hPSCs to mesendoderm (ME), mesoderm (MS), early cardiac (EC) cells and cardiomyocytes (CM). Cells were coaxed with physiological factors such as TGF-β ligands and bone morphogenetic proteins (BMPs) in the absence of serum. Design-of-experiment methods were implemented to determine combinations of factors, their concentrations and length of cell treatment after measuring the expression of specific markers at each stage. Differentiating hPSC progeny was analyzed by quantitative reverse transcription-PCR, immunocytochemistry, western blotting analysis and flow cytometry. Besides static cultures, hPSCs were also seeded on microcarriers and expanded in stirred-suspension bioreactors as described2,3. The concentration, viability and potential lysis of cells cultured in stirred suspension were determined.
Human PSC monolayers exposed to activin A in serum-free medium gave rise to ME cells as we reported2 resulting in the expression of brachyury (T) by over 90% of the cells after 1 day. Further exposure of ME cells to activin and BMP resulted in upregulation of MS markers including FLK1. Commitment to EC cells expressing NKX2.5 and GATA4 was achieved by inhibition of the canonical Wnt pathway. After 2 weeks of differentiation, cells emerged exhibiting heart cell markers such as cardiac troponin I (cTnI) and α-actinin and organizing into beating foci responding to drugs in a chronotropic manner.
Translation of this differentiation scheme to a scalable microcarrier bioreactor culture of hPSCs was further explored. Operating conditions such as the agitation rate, seeding cell density and cell/microcarrier ratio were selected for the cardiogenic differentiation of hPSCs. After expansion under non-differentiating conditions, cells were treated with the same protocol described above for static cultures of hPSCs. The differentiating cells expressed stage-specific markers in an orderly fashion and eventually formed clusters with contractile activity. Beating activity was modulated organotypically upon treatment with diltiazem and phosphodiesterase inhibitors. The final fraction of cardiomyocytes was determined by the expression of cardiac markers such as myosin heavy chain and cardiac troponins via flow cytometry. Approximately 7 cardiomyocytes were generated in the bioreactor culture per stem cell seeded compared to ~3.5 cardiomyocytes/hPSC in dishes. Ongoing efforts focus on optimizing the final concentration of hPSC-derived cardiomyocytes in stirred-suspension microcarrier cultures.
Our results demonstrate the stirred-suspension cultivation of hPSCs and their en masse differentiation along heart cell lineages in serum-free media with physiologically relevant factors. These findings support the development of bioprocesses based on this bioreactor system for producing clinically-relevant quantities of cardiomyocytes.
1. Jing, D. et al. Cell Transplant. 2010, 19:1397-412.
2. Lock, L.T. et al. Tissue Eng. Part A 2009, 15:2051-63.
3. Kehoe, D. et al. Tissue Eng. Part A. 2010, 16:405-21.