(122a) Expansion of Human Pluripotent Cells in a Microcarrier Bioreactor and Their Directed Differentiation Toward Pancreatic Islet Cells

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), which are collectively termed human pluripotent stem cells (hPSCs), hold great promises as sources of therapeutics for a wide range of maladies such as Parkinson disease, myocardial infarction and diabetes mellitus. Of major importance for hESC-based therapies to become a clinical reality is the development of bioprocesses for the expansion of stem cells and their derivatives to large quantities.

Stirred-suspension bioreactors are scalable and allow for continuous monitoring and control of the culture conditions. In this study, we investigated the growth and directed differentiation in microcarrier stirred-suspension bioreactor cultures of hPSCs. We demonstrated the use of a microcarrier stirred-suspension culture system for the propagation of pluripotent hPSCs. The effects of major operational variables such as the agitation rate and seeding cell density on hPSC growth and viability were investigated leading to the identification of optimal conditions for hPSC expansion. Stem cells seeded on microcarriers and cultivated for about one week in a stirred-suspension bioreactor remained viable (>85%) and increased 39.5±5.5-fold. The cells maintained their expression of pluripotency markers OCT3/4A, NANOG, TRA-1-81 and SSEA4 as revealed by quantitative PCR (qPCR) and immunostaining.

Beyond the propagation of undifferentiated hPSCs, we set out to develop a strategy for directing their fate along pancreatic islet cell lineages in bioreactor cultures. A detailed analysis was carried out to identify factors, which are involved in embryonic pancreas development such as activin, fibroblast growth factors (FGFs), and Wnt ligands, inducing the differentiation of hPSCs through the definitive endoderm (DE), primitive gut tube (PGT), and posterior foregut (PFG) stages. Cells emerged expressing biochemical and morphological markers characteristic of each stage. The concentration and time of addition of soluble differentiation factors to the culture as well as the duration of cell exposure to the stimuli were investigated. Selection of such conditions was based on the expression of appropriate markers in each stage and the final fraction of insulin-positive cells. The fraction of insulin-transcribing cells derived from hPSCs was assessed with the help of an adenoviral vector carrying a dual cassette encoding a reporter gene flanked by the insulin promoter, flow cytometry and immunocytochemistry.

Our differentiation protocol was employed in directing the fate of hPSCs in a microcarrier bioreactor culture. The cells transitioned through DE and PGT to PFG. Further differentiation of PFG cells on microbeads induced the expression of pancreatic islet markers such as insulin, PDX1, NKX2.2 and NGN3 as assessed by qPCR, immunocytochemistry and flow cytometry. Approximately 5% of the resulting population expressed insulin. Current efforts focus on optimizing the generation of beta-cell-like cells in this culture modality. We provide a first account of the expansion of hPSCs and directed differentiation into pancreatic progeny in a scalable microcarrier bioreactor. Our findings warrant further improvement in the efficiency of pancreatogenic differentiation of hPSCs. Moreover, the results illustrate the great potential of stirred-suspension bioreactor?based scalable bioprocesses for producing therapeutically useful cells from stem cells in clinically relevant quantities.