(805g) Engineering of Xeno-Free Microcarriers for the Scalable Expansion and Differentiation of Human Pluripotent Stem Cells in Stirred-Suspension Vessels

Human pluripotent stem cells (hPSCs) hold great promise as potential sources of cellular material for regenerative medicine and tissue engineering. Successful application of stem-cell based therapies however, will require the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable hPSC expansion and directed differentiation due to their simple operation, control of the culture environment and wide adoption by the pharmaceutical and biotechnology industries [1, 2, 3]. Comparative analyses of microcarriers available in the market clearly show the need for engineering synthetic substrates supporting the adhesion and growth of hPSCs in 3D cultures under agitation-induced shear.

We report the successful adaption of hPSC culture to peptide-conjugated microcarriers in xeno-free stirred-suspension bioreactors. Initially, the low microcarrier seeding efficiencies, which pose a bottleneck in the process, were improved by employing a newly developed protocol. Human PSC seeding fractions increased from 30% to over 80% and the duration of microcarrier loading phase was shortened substantially (to 4 hours from one to several days). Importantly, this method was combined with the generation of microcarriers featuring a vitronectin-derived peptide, which was shown previously to support the growth of human embryonic stem cells (hESCs). Peptide conjugation was achieved through an optimized ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS)-catalyzed amidation reaction. Human PSC adhesion was quantified against different peptide concentrations on the microcarrier surface. Peptide-conjugated microcarriers promoted hPSC attachment and growth under static conditions but failed to support proper growth under agitation-induced shear. After additional treatment of microcarriers with a synthetic substrate, human ESCs and induced PSCs (iPSCs) were successfully expanded under xeno-free conditions over multiple passages. Cell concentration increased 20- to 30-fold over each 6-day passage, viability remained above 85% and a normal karyotype was maintained. Furthermore, cultured cells retained the expression of pluripotency markers such as NANOG, OCT4 and SSEA4 analyzed by qPCR, flow cytometry and immunostaining. After expansion, the cells were released from the microcarriers and transferred to static cultures for differentiation. When subjected to spontaneous differentiation in embryoid body (EB) cultures or directed differentiation to the three embryonic germ layers, the cells adopted respective fates displaying relevant markers.

The use of these peptide-conjugated microcarriers was also explored for directing hPSC fate to specific progeny in stirred-suspension vessels. Expansion of hiPSCs on microcarriers was integrated with their directed differentiation to mesoderm progeny. The expression of Brachyury (T), KDR, MEOX1 and other markers was confirmed by RT-PCR, immunostaining and flow cytometry. Over 50% of the cells in xeno-free microcarrier bioreactors were KDR⁺compared to approximately 40% in dish cultures.

Thus, we demonstrated a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable, xeno-free propagation and differentiation of human stem cells intended for therapies.

Acknowledgements: National Institutes of Health (NHLBI): R01HL103709, and New York Stem Cell Science Trust (NYSTEM): contract C024355.

References:

1. Kehoe DE, Jing D, Lock LT, Tzanakakis EM (2010) Scalable Stirred-suspension Bioreactor Culture of Human Pluripotent Stem Cells. Tissue Eng Part A 16: 405-421.

2. Lock LT, Tzanakakis ES (2009) Expansion and differentiation of human embryonic stem cells to endoderm progeny in a microcarrier stirred-suspension culture. Tissue Eng Part A 15: 2051-2063.

3. Jing D, Parikh A, Tzanakakis ES (2011) Stem cell Bioprocessing for Regenerative Medicine. In:  Stem Cells: From Mechanisms to Technologies. Eds. Stachowiak MK, Tzanakakis ES. New Jersey: World Scientific. pp. 197-229.