(23f) Engineered Peptide Modified Hydrogel Platform for Biomanufacturing of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have the potential to revolutionize healthcare and have the capacity to differentiate into all mature cell types, making them attractive candidates for cell therapy based regenerative medicine. However, such commercial and clinical applications of hPSCs will require its mass production, necessitating an integrated, robust bioprocess for its scalable culture. Current approaches utilize suspension culture of hPSC aggregates, which maintains cell-cell contact and is highly scalable. However, controlling aggregate size, multi-aggregate coalescence, and hydrodynamic shear stress when cultured in a bioreactor is difficult. Hence it is challenging to control the heterogeneity in hPSC propagation, which is reflected in heterogeneity in hPSC differentiation, thereby compromising its functional maturation.

In contrast to the current approaches, we propose to develop a transformative approach which allows high viability and controlled propagation of single cell hPSCs. We hypothesize that ensuring single cell survival of hPSCs will be critical for its homogenous propagation. In addition, propagating the hPSCs within hydrogel capsule will protect the cells from bioreactor hydrodynamics, thereby ensuring controlled scale-up. We are thus designing peptide-conjugated alginate capsules to meet the biomanufacturing need of hPSCs. The designed peptides will ensure single cell survival and propagation, while alginate capsule will protect from bioreactor hydrodynamics.

hPSC survival and proliferation critically depends on the tight cell-cell contact, the absence of which results in ROCK pathway activation, actomyosin hyperactivation, and ultimately dissociation induced apoptosis. We have designed synthetic peptides which mimics cell-cell contact in hPSCs, promoting cell viability and pluripotency. Synthetic peptides which mimic the bioactive regions of full proteins are low cost and easily produced. Four synthetic peptides were designed and were first evaluated in 2D planar alginate substrates. hPSCs attached to the peptide modified alginate, and attachment increased as function of peptide concentration for each of the four peptide conjugated substrates. Substrates supported single cell attachment and viability of hPSCs in planar culture. Single cell hPSCs grew into colonies upon continued culture on modified substrates, supporting hPSC propagation. After propagation, hPSCs retained high levels of pluripotency comparable to parallel cultures on Matrigel, confirmed by high levels of OCT4 and Nanog gene and protein expression. Maintenance of pluripotency was dependent on peptide length and sequence, and in cases showed higher expression compared to Matrigel controls. Additionally, hPSC retained differentiation potential after propagation in the peptide conjugated alginate, comparable to cell differentiated on Matrigel. Cells were chemically induced to the definitive endoderm stage, and showed high levels of SOX17 and FOXA2 gene expression. Similar to propagation, differentiation was dependent on peptide length and sequence. In parallel, we have also determined properties of alginate capsules to support hPSC propagation and differentiation. We are currently integrating the identified peptides with the alginate capsules, to facilitate culture in the bioreactor setting. This will allow for large scale biomanufacturing of hPSCs within the hydrogel with high viability, while protecting hPSCs from aggregation or shear stress.