Ultrastucture Of Optic Vesicles-like Induced From Human Embryonic Stem Cell And The Primary Exploration Of Tissue Engineering Retina

The visual damage caused by retinal degenerative diseases,such as Age-related Macular Degeneration and Retinitis Pigmentosa is irreversible. It mainly involves photoreceptors and the retinal pigmented epithelial cells. Their deterioration causes patients’blindness. There is yet no effective methods to prevent disease progression and recover visual acuity. Gene therapy and antiangiogenesis therapy can only delay the progression of the diseases. The cell replacement therapy is to realize diseased retinal regeneration by transplantion of different kinds of cells, such as embryonic stem cells, retinal stem cells, induced pluripotent stem cells. But there are many problems of cell replacement therapy, including low survival rate and hard integration with host cells. With the development of tissue engineering, scientists begin to consider tissue engineering as a method of promoting the survival, differentiation and integration of transplanted cells. We construct tissue engineering of retina as the purpose of this topic and start with induction and differentiation of human embryonic stem cells. We provide reference with transplantation in vivo by studying the result of induction and differentiation, the scaffold construction of PLGA and the composite of "retinal cells-scaffold" in vitro culture.Retinal cells and retinal pigmental epithelia cells can be induced from human embryonic stem cells by the existing induction scheme. Directional differentiation can be realized by changing components. Cell types can be identified by immunohistochemistry and observe ultrastructure of cells by transmission electron microscope to judge whether they possess mature function. We fabricate PLGA scaffold by solvent casting and transplant retinal cells as seeding cells onto PLGA scaffold. After construction of composite of "retinal cells-scaffold", we observe the growth and development of cells and analyse the materials and structure of scaffold.Human embryonic stem cells are cultured in vitro and induced for 7 weeks. The clones of human embryonic stem cells have smooth edge and bright color. The cellular nucleo-cytoplasmic ratio is high and evenly distributed. The differentiated clones have a flat shape with color deepen in the centre and a lower nucleo-cytoplasmic ratio. The differentiated cells undergo embryonic bodies, rosette、retinal progenitor cells and retinal cells or retinal pigment epithelium cells. The results of immunohistochemistry show that hESCs are successfully differentiated into Chx10+ retinal cells、Mitf+ retinal pigment epithelial cells and mixed cells.Cells from different cell lines induced 7.5weeks to 13 weeks are observed finding cavity in most cell balls liking ultrastructure of optic vesicles-like. A few cells are division or apoptosis, which is similar to development model of neurepithelium in vivo. At an early stage of differentiation, cell arranged densely and cavity between cells can be observed. Cell nucleus are circular or irregular in shape. Nucleolus and heterochromatin are clear. Cells have short protrusions and few cytoplasm. At later stage of differentiation, there are a variety of cell shapes and cells near to cavity are long column, finding stuctures of cilium-like and membranous disc-like occasionally, which hints photoreceptors in differentiation. Cells differentiate obviously and cytoplasm increases, containing many organelle such as rough surfaced endoplasmic reticulum and mitochondria. Structure of cell connection-like and synaptic vesicle-like can be finded. Many melanin granules can be observed in cells close to cavity. Granules vary in size and colouration, which point out different development stage. There are tightly packed structure of microvilli-like in the free surface of the cell.Two kinds of porous polymer scaffolds are made, that is orientated scaffolds and non-orientated scaffolds. The scanning electron microscope show porous maximum diameter is about 100μm and porous communication by 25μm diameter. We cut the polymer into about 1mm thick and 5×5mm size. HESCs differentiated for 7 weeks are transplanted onto scaffolds by an orde of magnitude of 106. The composite are cultured for 2 days and observed by scanning electron microscope. The results show there is little cells on orientated scaffolds,while cells on non-orientated scaffolds have a high density and good growth state. Cells extend protrusions and connect with each others widely. This phenonmenon may be related to macropore. The pores run through scaffolds leading to cells leaking a lot Although macropore, the non-orientated scaffolds can carry a large number of cells and there is no phenonmenon of leaking. In addition, we can find two kinds of circumstances of cell distribution. One kind of cells is sheet in distribution similar to the retinal pigment epithelial cells. Another kind of cells form groups. It may be related to seeding cells including different kinds of cells.Retinal cells and retianl pigment epithelial cells can be successfully gained by existing scheme. The differentiated cells possessing the characteristic of retinal cells have pigment granules and disc membrane, etc. Cell junction identifies mature function of differentiated cells. The PLGA scaffold made by solvent-casting technique are biological compatibility. After transplantation by an orde of magnitude of 106 onto scaffolds of 1mm thickness and 5×5mm size, we can find two kinds of cell distribution, good growth state of cells and wide connection. Non-orientated scaffolds are superior to orientated scaffods duing to carrying more cells.