Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method
Authors
Heidi Hongisto , Tanja Ilmarinen, Meri Vattulainen, Alexandra Mikhailova and Heli Skottman
Institution
BioMediTech Institute, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland
Country
Finland
Year
2017
Journal
Stem Cell Research & Therapy
Abstract
Background: Human pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy,
but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and
feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target
cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs).
Methods: Multiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of
Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells
were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation
methods shared the same basal elements, using only minor inductive modifications during early differentiation
towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent
differentiation experiments and recovery after xeno-free cryopreservation.
Results: The defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs
with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation
had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed
functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as
phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC
differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established
xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful
recovery after thawing.
Conclusions: We propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs.
The two clinically relevant cell types are generated with simple inductive modification of the same basalmethod,
followed by adherent culture, passaging, and cryobanking.