| Backgroud: Retinitis pigmentosa (RP) is a kind of hereditary disease characterized by apoptotic degeneration of photoreceptors. So far, there is no curable method for RP.Transplantation of stem cells or retinal pigment epithelium has become the most promising way to treat RP. Retina is the immune-privilege organ. Cell transplantation via vitrectomy is a mature operation. In addition to the noninvasive observation after transplantion, eye has become the idealest organ to perform cell transplantational research. Restricted to ethical problems, the progresses of clinical and basal researches related to isolating stem cells directly from human tissues are slow.Owning the ability to perform trilaminar embryonic differentiation, human embryonic stem cells (hESCs) have become a type of ideal seed cells. In-vitro inducement of hESCs to form the organoids provided a good solution for the source of transplantational cells.Simultaneously, as the formation of organoids well repeats the organ-formation process during embryonic development, three-dimensional (3D) inducement of hESCs has become an excellent model for human development study. Currently, 3D inducement of hESCs is able to form different types of organs including optic cups. However, there are several problems of 3D inducement of hESCs to form neural retina (NR) need to be solved before further application: 1. There are still differences between in-vitro 3D formed NR and in-vivo generated NR. 2. Factors influencing the NR formation are not clear. The inducement rate is relatively low. Besides, as the cellular components within developing NR are complex, the location and isolation of retinal progenitor cells (RPCs) are formidable. In this way, selecting a better way to improve 3D culture as well as finding a proper membranous marker to isolate transplantable cells have become the research focus.Oxygen is essential for the presence of life. On the one hand, the oxidative phosphorylation of oxygen provides enormous ATPs which guarantee the efficiency of life activities. On the other hand, development of organism is accompanied by the variation of environmental oxygen concentration. It is generally believed that hypoxia is a mechanism for stem cells to maintain self-renew and sternness. And hyperoxia usually promotes the differentiation of stem cell. The 3D formed NRs are usually completed under normal oxygen concentration. However, the embryonic development of the central nervous system is preceded by the development of the cardiovascular system, which means that retina will experience a shift of oxygen concentration during the establishment of circulation system.Currently, lots of 3D inducements don't involve with this process.In addition, ideal transplantable cells should be qualified with following ablitities: 1.Being in the progenitor stage, which is convenient for in-vitro amplification and replacing the damaged cells after transplantation. 2. Non-embryonic origin, which guarantees the disablility of teratoma formation. Organ-derived Tyrosine-protein kinase Kit (C-Kit)-positive cells have the ability for self-renew, multicellular differentiation. Transplantation of C-Kit positive cells was able to repair the damaged cells. Whereas the stage-specific embryonic antigen 4 (SSEA4) is a surface marker of the human embryonic stage. SSEA4 is a membranous marker to distinguish cells with embryonic origin.Objectives: To investigate the cell and tissue structure of 3D induction of hESCs formed NR. To explore the effect of oxygen concentration on the development of NR and futher improve 3D inducement via controlling the oxygen concentration. To study the biological characteristics of C-Kit+/SSEA4- cells isolated from the induced NR as well as the effect of hypoxia on the C-Kit+/SSEA4- cells and further harvest RPCs in good viability and abundant cell amount. To analyze the features of gene expression of the C-Kit+/SSEA4- cells by transcriptome scanning and further thoroughly understand the RPCs.Methods and results:The study is divided into three parts:Part 1: 3D culture of hESCs and spontaneous formation of NR1. Using cell immunofluorescence, flow cytometry to study the proliferation characteristics and embryonic origin of hESCs cell line H1. It was found that H1 cells were cloned and aggregated. The mitotic phases of H1 cells were obvious. Over 95% H1 cells expressed the embryonic markers.2. Using immunofluorescence staining to study 3D induction of hESCs to form visual vesicles and the optic cup-like tissue. It was found that the visual vesicles and be viewed at about 8-day culture. When cultured 24 days, the shape of the optic vesicle was more clear and double-layer structure could be observed. 38-day inducement of NR could express the proliferation marker: Ki67; the RPCs markers: PAX6, RAX and CHX10; the neural progenitor markers: SOX2 and NESTIN; the retinal ganglion cell marker: Tuj1 and the photoreceptor marker: Crx. RAX was expressed in all NR cells.Part 2: The effects of oxygen concentration on the development of NR formed by 3D inducement of hESCs1. Using immunofluorescence, the effect of high oxygen concentration on the formation of NR from hESCs was studied. It was found that hyperoxia could significantly promote the development of NR and its cell proliferation. Besides, the migration of proliferating cells between apical-basal sides was enhanced, which meant that hyperoxia could promote the interkinetic nuclear migration within NR.2. Using immunofluorescence, the apical-basal polarity of neural rosettes within NR was studied. It was found that the apical side of neural rosettes would turn over in 20%O2 groups. Neural rosettes could develop with normal apical-basal polarity in 40% O2 group.3. Using immunofluorescence, the effect of oxygen concentration of the development of RPCs within NR was studied. It was found that the shift of oxygen concentration did not change the expressing pattern of retinal markers. However, the generation of PAX6-positive RPCs was increased under high oxygen concentration (40% O2), compared to normal oxygen concentration (20% O2). And hyperoxia significantly promoted the formation,migration and differentiation of ganglion cells.Part 3: Oxygen concentration influence the hESCs 3D neural retina-derived C-Kit+/SSEA4- cells and its biological characteristics1. Using improved way to induce hESCs to form NR, the general characteristics of oxygen concentration and the expression of nerve retinal markers were studied. It was found that the improved inducement of embryoid body was relatively slow. The cell and tissue structure were not changed. Growth could be promoted by high oxygen.2. Using immunofluorescence staining to study the distribution of C-Kit protein. It was found that the C-Kit positive cells were mainly distributed in the inner layer of the NR and expressed Nestin, PAX6 and RAX, but not CHX10. C-Kit expression was declining with time went on.3. The status and proliferation of hESC-NR-C-Kit+/SSEA4- RPCs were studied by cell viability assay. The hESC-NR-C-Kit+/SSEA4- proliferated better under hypoxia. Sorting cells on 30 days, 45 days and 60 days showed that the proliferation reached highest in 30-day isolated cells.4. Using immunofluorescence staining, the characteristics of hESC-NR-C-Kit+/SSEA4- cells were analyzed. The results showed that hESC-NR-C-Kit+/SSEA4- cells could express the various RPCs markers including Nestin, PAX6 and RAX. Besides, hESC-NR-C-Kit+/SSEA4- cells could be induced to differentiate into ganglion cells, bipolar cells,photoreceptors and Muller cells.5. The gene expression of hESC-NR-C-Kit+/SSEA4- cells and human RPCs were studied by using genomic transcriptional analysis. The results showed that all 30 days, 45 days and 60 days sorted hESC-NR-C-Kit+/SSEA4- cells were very close to RPCs isolated from human embryos with related factors over 0.88. Besides, 30-day isolated cells had the highest factor: 0.908. But gene expression related to proliferation and migration was increased in hESC-NR-C-Kit+/SSEA4- cells. While signaling pathway relating the cell adhesion moleculars was inactivated in hESC-NR-C-Kit+/SSEA4- cells.Conclusion:1. 3D inducement of hESCs formed NR could express various RPCs markers. The development of in-vitro induced NR was similar to the in-vivo generated NR. The improved 3D culture (BMP4 conducted) method was in a relatively simple process with less additives,which was more proper for future clinical application.2. The effects of oxygen concentration on the formation of three-dimensional NR included: promoting the proliferation of NR; promoting the development of neural rosettes in the physiological apical-basal model; promoting the formation and migration of PAX6-positive RPCs; promoting the formation, maturation and migration of ganglion cells. All these results illustrated that high oxygen concentration is helpful to harvest more NRs closer to in-vivo development.3. Under hypoxia (3%), the cell viability and proliferation activity of sorted hESC-NR-C-Kit+/SSEA4- cells was significantly improved, which showed that hypoxia was in favor of stabilization and amplification of seed cells.4. C-Kit positive cells could simultaneously express Nestin, PAX6, RAX but not embryonic markers. Sorted hESC-NR-C-Kit+/SSEA4- cells were able to differentiate into subretinal cells including ganglion cells, bipolar cells, photoreceptors and Muller cells. All these results showed that hESC-NR-C-Kit+/SSEA4- cells were a subtype of RPCs.5. Transcriptome study proved that hESC-NR-C-Kit+/SSEA4- cells sorted from the induced NR were close to RPCs isolated from human eye (adult RPCs) (>90%). However,the proliferation and migration of hESC-NR-C-Kit+/SSEA4- cells were better than adult RPCs. These results indicated that hESC-NR-C-Kit+/SSEA4- cells were a type of RPCs cloest to in-vivo development, which brought a new seed cell source with low tumor formation risk, good differentiation potential and standardized ability for stem cells treatment of RP. |
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