טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentLiat Aharon
SubjectDifferentiation of Human Embryonic Stem Cells into
Retinal Cells
DepartmentDepartment of Biotechnology
Supervisors Professor Emeritus Perlman Ido
Professor Emeritus Itskovitz Joseph
Full Thesis textFull thesis text - English Version


Abstract

The neural retina is subject to a number of degenerative diseases, including retinitis pigmentosa, age-related macular degeneration (AMD) and glaucoma.
 Many of the retinal degeneration diseases in human are caused by impairment in the photoreceptor cells. Therefore, in vitro generation of photoreceptors offers an exciting potential for future therapeutical intervention. Human embryonic stem cells (hESCs) are undifferentiated cells derived from the inner cell mass of blastocysts. The cells can be cultured for prolonged periods, while remaining undifferentiated and maintaining a stable karyotype. The cells also maintain their potential to differentiate into derivatives of all three germ layers both in vitro and in vivo.

The aim of this research was induction of hESCs differentiation into retinal neurons, with particular emphasis on photoreceptors. The key to obtain maximal retinal lineage commitment, from hESCs, involves identifying the correct combinations of molecular cues to optimize efficacy of target cell derivation. Differentiation was induced using several culture systems. In the first two culture systems we started with a two stage approach. The first stage was establishing an enriched culture of hESCs-derived Neural Progenitors (NPs). Next we attempted to evaluate the affect of combinations of different feeders and factors, upon the differentiation of NPs towards retinal progenitor and photoreceptor cells.

Then we examined a direct differentiation system, which did not involve generation of neural spheres. In order to determine the ability of RPE cells to induce hESCs to differentiate along retinal progenitor and photoreceptor lines, undifferentiated hESCs were cultured on ARPE-19 feeder layer in the presence of serum free medium, or on matrigel RPE-CM.

We managed to establish an enriched culture of NPs that expressed markers of retinal differentiation lineage from hESCs in vitro, using different cultures. These NPs can be used for the analysis of early development, and as groundwork for the differentiation of hESCs into the various cell types of the nervous system, and into retinal progenitor cells. Our differentiation systems and the resulting cells may be useful for cell therapy, for testing drugs and for studying the development of the human central nervous system, especially the eye.