Expansion and Neural Commitment of Human Pluripotent Stem Cells As 3D Suspension Aggregates

Human pluripotent stem cells (hPSC) hold great promise to enable cell therapies, tissue regeneration and a better understanding of embryonic development and cell differentiation. Though great advances have been made in the study of these cells, their translation to the clinic is being hampered by technical issues, particularly related with their large scale expansion and the efficiency of the in vitro differentiation protocols. Therefore, more efficient and cost-effective methodologies need to be developed.

In order to better mimic the in vivo microenvironment, 3D suspension conditions are starting to emerge as a promising alternative to perform the efficient in vitro expansion and controlled differentiation of hPSC. This culture system is based on the formation of cell aggregates initiated by single-cell suspensions, manually dissociated cell clumps or cultured in microwells that allow the formation of aggregates with an homogeneous and controlled size distribution. These culture systems were evaluated and compared in this work to perform the expansion and neural commitment of hPSC lines.

When cultured in the presence of expansion medium, hPSC aggregates derived from single-cell suspensions exhibited a narrow range of size distribution after seven days of culture whereas manual dissociation resulted in a wider range of diameters. To facilitate the study of the effect of aggregate size in the expansion of hPSCs, aggregates ranging from 200 to 5,000 cells per aggregate were formed by changing the initial cell concentration, and forcing the cells to aggregate in AggreWell™ plates. After one week in culture, the fold increase in total cell number changed in an inverse proportion to the aggregate size and, therefore, 200 initial cells per aggregate reached the highest value (16.0±2.7). Manually dissociated aggregates achieved intermediate fold increase values (3.8±1.7), possibly due to aggregate heterogeneity. After three weeks in culture, cells exposed to the three different conditions were analyzed for the expression of pluripotency markers by flow cytometry and at least 90% of the cells were found to retain the expression of OCT4 and TRA-1-60. 

Several methodologies have been applied to induce the neural commitment of hPSC, including the Neural Sphere [Steiner et al., 2010] and the Dual Smad Inhibition [Chambers et al., 2009] methods. Nevertheless, their performance has never been compared side by side. Since the neural sphere induction method requires cells to be cultured for four weeks, the growth of the aggregates is limited to the point where their diameter will condition the diffusion of nutrients, oxygen and metabolites to and from the inner volume of the aggregate. As a result, aggregates with smaller initial sizes, namely 200 and 1,000 cells per aggregate, were shown to lead to higher cell yields (7.6±0.15 and 13.1±1.4, respectively), whereas the final expression of OCT4 for all sizes of aggregates remained below 3% after neural specification. On the other hand, the dual Smad Inhibition method, initially designed to induce neural commitment in monolayer cultures, appears to require a higher initial cell density, attaining higher cell yields when using 5,000 or 10,000 initial cells per aggregate (5.0±0.4 and 6.6±0.5, respectively). Also, when using N2B27 chemically-defined neural induction media supplemented with LDN193189 and SB431542 small molecules, instead of the traditional Knockout serum replacement (KOSR)-supplemented medium, it was possible to decrease the percentage of OCT4-expressing cells from >95% at day 0 to ~30%, after twelve days of neural commitment. Moreover, after replating the cells it was possible to give rise to neural rosette colonies that were tested positive for the expression of the neural markers NESTIN, PAX6, FoxG1 and SOX2, confirming that this process is also suitable to induce hPSC neural commitment in a 3D suspension culture.

References

Chambers, S.M., Fasano, C.A., Papapetrou, E.P., Tomishima, M., Sadelain, M.,Studer, L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275-280 (2009).

Steiner, D., Khaner, H., Cohen, M., Even-Ram, S., Gil, Y., Itsykson, P., Turetsky, T., Idelson, M., Aizenman, E., Ram, R., Berman-Zaken, Y.,Reubinoff, B. Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat Biotechnol 28, 361-364 (2010).