| Age-related cataract (ARC) is the main cause of blindness worldwide. There are about2.5million blind people who was caused by cataract in China, accounting for10%of global cataract-induced blindness. With the aging of the population, the expected annual increase of cataract patients will be more than one million, which far exceeds the current surgery load. Therefore, it is a great challenge to prevent and treat the cataract-induced blindness in China. Congenital cataracts are caused by genetic or developing disorder which may exist premature or develop gradually after the birth. More than1/3congenital cataracts associated with genetic problems. Any mutation of genes which paticipate in or control the development of lens may lead to the congenital cataracts. Therefore, it is of great significance to treat and prevent the development of congenital contaract to clarify its pathogenesis. Cataract extraction with intraocular lens (IOL) implantation is the ideal and most commonly used approach of cataract treatment. However, posterior capsular opacification (PCO), the most common complication which may aggect the long-term prognosis of visual acuity occurs in about30%cateract patients in the first5years postoperatively. Although this complication can be treated by laser or surger, it will increased the economic burden on the patient and will bring other new complications. So how to prevent PCO causes ophthalmologists’ and patients’ great concern.Inducing somatic cells to pluripotent stem cells was a great innovation and milestone in bioscience. Induced pluripotent stem cells (iPSCs) can self-renew and differentiate into almost all cell types in vitro and in vivo, which have great application potential in regenerative medicine and high vaule in theoretic research. IPSCs obtained from embryonic stem cells in the inner cell mass of blastocysts, the nuclear transfered embryonic stem cells and the exogenous transcription factor transferred somatic cells were used to conduct the somatic cell reprogramming research. In just a few years, this study has achieved many breakthrough advancement in fields of cell reprogramming research, pathogenesis research and clinical application. This non-cloning stem cell technology managed to avoid the long time debating ethical issues, greatly promoted the development of this technology and related sciences. Compared with the embryonic stem cell research, somatic cell reprogramming technology can supply pluripotent stem cells to more patients. Thus, it has broad prospects in medical applications (such as organ culture, and organ transplantation, etc.).Establishment of patient-specific pluripotent stem cells can provide a powerful tool for the study of disease pathogenesis, treatment, drug screening and cell transplantation therapy. Exogenous transcription factor was transferred into the lens epithelial cells of contaract patients by lentiviral vector and then the induced pluripotent stem cells were acquired. The establishment of iPSCs from cataract patients can provide a convenient cell model for lens development, cell differentiation and aging. Meanwhile, it will provide a powerful research tool for anti-contaract drug screening and pathogenesis research.Part I Culture of primary human lens epithelial cells in vitroObjective The lens epithelial cells from normal peopole and age-related cataract patiens and human dermal fibroblasts were cultured in vitro. The biological characteristics and histological changes of the in vitro cultured lens epithelial cells and dermal fibroblasts were observed.Methods Lens capsure from keratoplasty donor eyes was derived for normal human lens epithelial cell culture and anterior lens capsule dilacerated in the phacoemulsification surgery was used for age-related cataract lens epithelial cell culture. The posterior capsure of keratoplasty donor eyes was cut along the circle2mm behind the lens equator and the anterior and equtorial capsure was completely removed carefully. Then the obtained capsure was cut into1mm×1mm square pieces. The tissue pieces were cultured in basks directly. The anterior capsure of cataract lens was completely obtained in phacoemulsification surgery and the viscoelastic agent was washed away. The capsure was also cut into1mm×1mm square piece and cultured in basks directly. The fused cells was digested and passed by0.25%trypsin solution. Skin tissue of the age-related cataract patients was obtained and cut into1mm3tissue blocks, digested overnight and cells were collected after centrifugation. After several passages, more purified skin fibroblasts were obtained and cultured. The morphology of these three types of cells were observed by inverted phase contrast microscope. The proliferation rate of these three cells was determinated by CCK-8assay for7days and the growth curve were compared among these cells.Results Human lens epithelial cells grew from the edge of the tissue block48to72hours after the attachment of the normal lens capsule and fused in10to15days. In the early stage, normal lens epithelial cells were polygonal, the cell body was bright and the cell boundary was clear. After the fusion, the cells had the morphological characteristics of epithelial cells and presented as the typical hexagonal or polygonal shape. Cells could be passed to the fifth generation in vitro, but after the third generation, the cell phenotype turned to fibroblast cells. After the third generation, normal human lens epithelial cell took a spindle or a long strip shape. The fifth generation of normal lens epithelial cells aged, vacuolar changes in different sizes could be found in the cytoplasm and finally collapsed to death. The cell morphology and proliferative capacity of most cataract lens epithelial cell were similar to those of normal lens epithelial cells, but some cataract lens epithelial cells were different in cell morphology from normal human lens epithelial cells, which were more similar to fibroblast cells and collapsed after the first passage. Human skin fibroblasts had spindle or polygonal shapes. The purified fibroblasts could be obtained after several passages. The growth curve drawn by the CCK-8assay showed that human skin fibroblasts had the highest proliferation rate and cataract lens epithelial cells had the slowest rate.Conclusion Human lens epithelial cells could be easily cultured by our method with a high success rate. The second generation of human lens epithelial cells was the ideal subject for cytology research and other related researches. Normal lens epithelial cells had good growth and proliferation capility and cataract lens epithelial cells had poor proliferation capility and poor cell morphology. After digestion and centrifugation, human skin fibroblasts could be purified by several passages. Human skin fibroblast proliferated fastest, normal human lens epithelial cell had a median proliferation rate and cataract lens epithelial cell proliferated slowest.Part Ⅱ Establishment of induced pluripotent stem cells from cataract patientsObjective Lentivirus vectors expressing OCT-4, SOX-2and KLF-4were respectively established and then lens epithelial cells from age-related cataract patients were transfected. We tried to induce and establish the induced pluripotent stem cells from patients and compare them with skin fibroblasts from the same patient. The inducing efficiency of lens epithelial cells and skin fibroblasts were observed.Methods Three patients diagnosed as age-related cataract were selected and the anterior lens capsule and skin sample of who were obtained for culture in vitro. The anterior capsure of cataract lens was completely obtained in phacoemulsification surgery and the viscoelastic agent was washed away. The capsure was also cut into1mm×1mm square pieces and cultured in basks directly. The fused cells was digested and passed by0.25%trypsin solution. Skin tissue of these three patients was obtained and cut into1mm3tissue blocks, digested overnight and cells were collected after centrifugation. After several passages, more purified skin fibroblasts were obtained and cultured.293T cells were infected by the OCT-4, SOX-2and KLF-4lentiviral vector plasmid (plenti-hOCT4/plenti-hSOX2/plenti-hKLF4) and the packaging plasmid (pCMV-gag-pol,-PA/pCMV-VSVg) respectively and then recombined and packed to construct lentiviral vector (Lenti-OCT-4/Lenti-SOX,2/Lenti-KLF-4). Three lentiviral vectors were mixed to infect the second generation of cataract lens epithelial cells and human skin fibroblast cells. After5-day culture, they were co-cultured with mouse embryonic fibroblasts (MEFs) until iPSCs appeared. The difference of iPSCs inducing efficiency between cataract lens epithelial cells and human skin fibroblast cells was compared by alkaline phosphatase (AKP) staining. Human H9embryonic stem cells (ESCs) were also cultured for comparision.Results Cataract lens epithelial cells and skin fibroblasts cultured showed good morphology and proliferation capility and the the second generation cells were suitable for lentiviral infection. These two kinds of cells could be transfected by the Lenti-OCT-4/Lenti-SOX-2/Lenti-KLF-4lentiviral vectors with high efficiency and the cell status and proliferative capacity were not affected. The cataract lens epithelial cells and human skin fibroblasts were co-cultured with MEFs5days after transfection. Cell clones which had the similar morphology with ESCs began to appear12days after transfection. AKP staining of cell clones was positive, suggesting iPSCs were successfully induced. The difference of iPSCs inducing efficiency between cataract lens epithelial cells and human skin fibroblast cells of these three patients was compared by alkaline phosphatase (AKP) staining. Under the1×106cell density, cataract lens epithelial cells can generate27.7AKP-positive cell clones while human skin fibroblasts only generated15cell clones. iPSCs cell clones induced from cataract lens epithelial cells had similarities in cell morphology with ESCs.Conclusions Cataract lens epithelial cells and human skin fibroblasts could be transfected by the Lenti-OCT-4/Lenti-SOX-2/Lenti-KLF-4lentiviral vectors with high efficiency and successfully induced iPSCs which was similar in morphology with ESCs and positive in AKP staining. Cataract lens epithelial cells had a higher iPSCs inducing efficiency than human skin fibroblasts.Part Ⅲ Idnetification of induced pluripotent stem cells from cataract patientsObjective The iPSCs from cataract patients were indentified by molecular biomarkers and in vitro and in vivo differentiation capacity, with the pluripotency of which was evaluated.Methods The pluripotency of the iPSCs derived from the cataract patients was identified by the following methods:gene expression, immunofluorescence staining analysis, RT-PCR analysis, in vitro differentiation of embryoid bodies and differentiated teratoma examination in vivo. Total RNA extracted from iPSCs, ESCs and cataract lens epithelial cells were analyzed for gene expression profiles detection by the HG-U133-2array (Affymetrix). Total RNA extracted from iPSCs, ESCs and cataracts lens epithelial cells was analysed by RT-PCR to detect the typical gene expression of human ES cell (Nanog, OCT-4, REX-1, SOX-2, VIMENTIN). The characteristic antigen expression of human ES cell (SSEA-3, SSEA-4, Nanog, TRA-60, TRA-81and OCT-4) were detected by immunohistochemical fluorescence (ICH). IPSCs were suspensedly cultured to observe the formation of embryoid body (EB) and special ingredients were added in the culture medium (BMP4, FBS, retinoic acid (RA)) to observed the in vitro differentiation of EB. The iPSCs were injuected subcutaneously into nude mice to observe teratoma formation and differentiation in vivo.Results The results of gene expression analysis showed that the gene expression profiles were very similar between the iPSCs and ESCs while they were quite different between the iPSCs and the cataract lens epithelial cells. We chose iPSCs from one56-year-old age-related cataract patients and randomly select four cell clones (named as iPS1, iPS2, iPS3and iPS4respectively) for examination. RT-PCR analysis showed that the characteristic gene of ESCs (Nanog, OCT-4, REX-1, SOX-2and vimentin) expressed similarly in PS1and ESCs while the other three clones expressed different from the ESCs. ICH examination of iPS1suggested characteristic antigens of human ES cell (SSEA-3, SSEA-4, Nanog, TRA-60, TRA-81and OCT-4) obviously expressed in iPS1. The result of in vitro embryoid body differentiation showed that the differentiation of EB downregulated the expression of Nanog and OCT-4. After BMP4, FBS and RA were added to the medium, the expression of characteristic genes of ectoderm (NACM, TH and GFAP), the characteristic genes of endoderm (SOX7and AFP), the characteristic genes of mesoderm (PECAM, desmin and SCL) upregulated in the EB at different levels. ICH test of EB cells showed that EB cells expressed the mesodermal marker α-smooth muscle actin (a-SMA), ectodermal marker beta-microtubule protein (Tuj-1) and the endoderm marker alpha-fetoprotein (AFP). In vivo teratoma differentiation showed that tumor formatted at the injection site one month after iPSCs injection. Two months later, the tumor was removed for tissue slice examination, the tissue cells of three germ layers could be observed in the tumor.Conclusion The iPSCs were similar with the ESCs in gene expression profile, ICH, RT-PCR results, in vivo and in vitro differentiation capacity. The iPSCs had the same pluripotent differentiation capacity with ESCs.Part Ⅳ Inducing differenciation of induced pluripotent stem cells from cataract patients to the lens progenitor cellsObjective To explore the three-step method, which could take advantage of the inducible factors to induce the iPSCs from cataract patients to lens progenitor cells and evaluate the similarity between differentiated cells and lens cells.Methods iPSCs induced from cataract lens epithelial cells and ESCs were further differentiated using a3-step induction procedure [6]:1) administration of100ng/ml Noggin (R&D) from d0to5;2) a combination of100ng/ml bFGF (R&D),20ng/ml BMP4(R&D) and20ng/ml BMP7(R&D) from d5to15; and3) a combination of100ng/ml FGF2(R&D) and20ng/ml Wnt-3a (R&D) from d15to30. The RNA was extracted from iPSCs and ESCs every5days and the expression of lens cell differentiation marker gene PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1and MIP were examined by RT-PCR and the curves were drawn. The expression of lens cell differentiation markers PAX6, α-crystallin protein and β-crystal protein was analysed by ICH. The expression levels of lens differenciation markers PAX6, a-crystallin protein and β-crystallin protein were compared by Western-blot in the total protein extracted from the iPSCs, ESCs and the lens protein. Total protein were extracted in three lenses from subjects aging22,44and65respectively and total protein of iPSCs from cataract lens epithelial cells, iPSCs from human skin fibroblasts and ESCs were also extracted. Connexin43and fibronectin were examined by Western-blot in order to evaluate the extent of epithelial-mesenchymal transition (EMT).Results After the3-step induction, the RT-PCR analysis of the differenciated iPSCs and ESCs showed that the expression of PAX6, CRYAB, CRYAA, BFSP1and MIP were increasing while the expression of SOX2was decreasing and the expression of SIX3started with increase and then decreased. ICH analysis showed that during the differentiation process, the lens differenciation markers PAX6, α-crystallin protein and β-crystal protein were continuously expressed in iPSCs. Western-blot analysis showed that PAX6, α-crystallin protein and β-crystallin protein were expressed in iPSCs, ESCs and lens, but the expression level in ESCs was relatively lower. EMT evaluation showed that connexin43expressed highest in the22-year-old lens and decreased significantly with aging while the fibronectin expression was gradually increased with age increasing; in cells differenciated from iPSCs from cataract lens epithelial cells, iPSCs from human skin fibroblasts and ESCs, the connexin43expressed highest in iPSCs from cataract patients and the expression of fibronectin were similar between groups.Conclusion The lens progenitor cells could be successfully induced by the combined effect of the3-step induction factors. The differentiated cells could stably express the characteristic gene and protein of lens progenitor cells. Compared with the iPSCs from human dermal fibroblasts and ESCs, iPSCs from cataract lens epithelial cells had a lowest EMT level. |
PDF Full Text Request