Abstract
Purpose: :
Retinal degenerative diseases are a group of largely untreatable clinical conditions in which the dysfunction and death of retinal photoreceptor cells lead to vision loss. Stem cell-based therapy appears as a promising treatment for this type of diseases. The purpose of this study is to develop improved methods to induce hiPS cells to differentiate into retinal cells and to test their potential to form tridimensional retina-like structures in vitro.
Methods: :
IMR90-clone 4 hiPS cells (WiCell Research Institute), were cultured and maintained on MatriGel with mTeSRTM1 medium according to WiCell feeder independent pluripotent stem cell protocols. The procedure for inducing differentiation of hiPS cells toward a retinal fate was based on a previously described protocol (Meyer et al. PNAS 2009) with some modifications. Differentiating hiPS cells were followed by morphological observation under inverted microscope every other day and characterized by immunocytochemistry with molecular markers of pluripotency and/or specific for different stages of retinal cell differentiation.
Results: :
Under specific differentiation conditions, hiPS cells lost expression of the pluripotency genes and gradually acquired expression of transcription factors specific for early stages of retinal cell differentiation. When grown in suspension, these cells form vesicular aggregates, morphologically resembling optic vesicle structures. Over time, these vesicular aggregates developed localized areas of pigmentation, highly resembling retinal pigment epithelium (RPE). Differentiating hiPS cells in the vesicular aggregates expressed markers characteristic of different stages of retinal cell differentiation, as well as of specific retinal cell types. Interestingly, the timeframe for acquisition of retinal cell fates is very close to that of human retina embryogenesis
Conclusions: :
Our results indicate that hiPS cells can be directed to differentiate into highly-organized cellular structures that closely resemble the early retina in both, structural organization and expression of cell-type specific markers. Studies to further characterize the level of differentiation achieved by hiPS cells in this system are currently underway.
Keywords: retinal development • differentiation • gene/expression