Scalable Xeno-Free Expansion of Human Pluripotent Stem Cells in Stirred-Suspension Vessels and Their Differentiation into Pancreatic Progenitor Cells
advances on human pluripotent stem cells (hPSCs), including human embryonic stem cells
(hESCs) and induced pluripotent stem cells (hiPSCs) have brought us closer to the
realization of their clinical potential. Nonetheless, tissue engineering and
regenerative medicine applications will require the generation of hPSC products well
beyond the laboratory scale. This also mandates ... read morethe production of hPSC therapeutics in
fully-defined, xeno-free systems and in a reproducible manner. In this work, we first
showed that hPSCs can be propagated on peptide-conjugated/poly-L-lysine (pLL)-treated
microcarriers in spinner flasks for long-term while maintaining their pluripotency,
normal karyotype, proliferation and differentiation capacity. Mesoderm differentiation
of cells can be integrated as a single process with expansion of hPSCs on the
microcarriers without compromising differentiation efficiency. Considering the specific
gravity of the peptide-conjugated microcarriers and the complexity in the preparation,
we developed another approach to engineer polystyrene microcarriers with vitronectin,
human serum albumin (HSA) and UV irradiation. In this way, we were able to prepare
xeno-free VN-HSA-UV microcarriers for long-term cultivation of hPSCs observing yields
comparable to those in cultures with peptide-conjugated microcarriers. Preparation of
the VN-HSA-UV beads however was significantly shorter and more straightforward. On the
other side, a differentiation protocol to coax hPSCs into insulin-expressing pancreatic
progenitor cells was developed under completely xeno-free conditions. All xenogeneic
components were eliminated or substituted with xeno-free reagents. The specification
strategy was optimized based on both the differentiation and the maintenance of cell
viability. In conclusion, this work provides a step forward to the development of
bioprocessing of hPSCs integrating expansion and directed pancreatic differentiation of
human stem cells for the production of therapeutically useful pancreatic cell
Thesis (Ph.D.)--Tufts University, 2016.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: Emmanuel Tzanakakis.
Committee: Qiaobing Xu, Kyongbum Lee, and Laertis Ikonomou.
Keyword: Chemical engineering.read less
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