Abstract
Purpose: :
Human induced pluripotent stem cells (hiPSCs) have received attention for their potential in the field of regenerative medicine. However, established methods for deriving retinal phenotypes from hiPSCs typically involve the use of feeder cells or other animal products. In order for hiPSCs to realize their therapeutic potential, it will be necessary to demonstrate the ability to derive mature phenotypes in the absence of animal products. Therefore, we sought to develop a simple method of deriving retinal cells from undifferentiated hiPSCs under xeno-free conditions.
Methods: :
hiPSCs were initially maintained through traditional methods on a layer of irradiated MEF cells, or through their growth in xeno-free medium on synthetic xeno-free Corning® SynthemaxTM Surface culture plates. After a minimum of 5 passages in a particular environment, cells were differentiated with minor modifications to our previous retinal differentiation protocol. Cells that had been maintained in xeno-free conditions were differentiated in serum-free media containing xeno-free media supplements. At various timepoints, cells were tested for the expression of markers of retinal development using immunocytochemistry and RT-PCR analyses.
Results: :
hiPSCs maintained in either traditional or xeno-free culture conditions were capable of repeated passaging and maintenance of a pluripotent state as determined by the expression of pluripotency genes. Upon differentiation in xeno-free conditions, cells acquired advancing features of retinal development, including cell populations analogous to the eye field and optic vesicle/cup stages of development. Furthermore, this differentiation in xeno-free conditions occurred in a manner similar to those cells differentiated using previously established protocols.
Conclusions: :
Results from this study demonstrate that hiPSCs can be maintained and directed to differentiate under xeno-free conditions similarly to previously established protocols using feeder cells and animal products. This capability will facilitate future efforts to develop hiPSC-based therapies for retinal disorders.
Keywords: retinal development • differentiation