Expansion and directed differentiation of human pluripotent stem cells to insulin-producing cells in a stirred-suspension microcarrier system.

摘要

Human embryonic stem cells (hESCs) are a promising source of therapeutics for diabetes patients. Of major importance for hESC-based therapies to become a clinical reality is the development of robust, directed differentiation protocols as well as of scalable bioprocesses for the expansion of stem cells and their derivatives to large quantities.;We first set out to develop a strategy for directing hESCs fate along pancreatic islet cell lineages in static cultures. A detailed analysis was carried out to identify factors, which are involved in embryonic pancreas development. The potential of activin, fibroblast growth factors (FGFs), and retinoic acid (RA) for the induction of hESC differentiation through the definitive endoderm (DE), primitive gut tube (PGT), posterior foregut (PFG) and pancreatic islet (PI) stages was investigated. Cells emerged expressing biochemical markers and morphology specific to 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 differentiation conditions was based on the expression of appropriate markers in each stage and the viability of cells. The fraction of insulin-transcribing cells derived from hESCs was assessed with the help of an adenoviral vector carrying a dual cassette encoding a reporter gene flanked by the insulin promoter, as well as quantitative PCR (qPCR), flow cytometry and immunocytochemistry.;In parallel, microcarrier stirred-suspension bioreactors, which are characterized by superb scalability, were investigated for the growth and directed differentiation of hESCs. We demonstrated the use of a microcarrier stirred-suspension culture system for the propagation of pluripotent hESCs. The effects of major operational variables such as the agitation rate and cell seeding density on hESCs growth and viability were investigated, leading to the identification of optimal conditions for their 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 in concentration. The cells maintained their expression of pluripotency markers OCT3/4A, NANOG, TRA-1-81 and SSEA4 as revealed by qPCR and immunostaining.;Subsequently, the differentiation protocol for directing the fate of hESCs was employed in the microcarrier bioreactor culture. The cells transitioned through DE and PGT to PFG at a similar efficiency with those in static cultures. Further differentiation of PFG cells on microbeads induced the expression of pancreatic islet markers such as insulin, PDX1, NKX6.1, NKX2.2, GLUT2 and NGN3. Approximately 8% of the resulting population expressed insulin in the bioreactor differentiation.;In summary, this dissertation describes the development of hESC directed differentiation into insulin-producing cells. Results from this work provided first account of hESC propagation and differentiation in a scalable microcarrier bioreactor. The great potential of stirred-suspension bioreactor--based scalable bioprocesses for the production of therapeutically useful cells from stem cells in clinically relevant quantities was illustrated.