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Objective:To establish a fluorescent reporter human induced pluripotent stem cell line (hiPSCs) for monitoring the expression of visual system homeobox 2 ( VSX2). Methods:VSX2_small guide RNA (sgRNA) was inserted into vector PX459 to construct knockout plasmid, and the P2A-eGFP knock-in donor plasmid was conducted at the same time.The two plasmids were transfected into BC1-hiPSCs.Single cell clones were generated after treatment of puromycin.Correct insertion was confirmed by PCR and Sanger sequencing.The isogenicity of the parental and the reporter hiPSCs was confirmed by STR analysis and karyotyping.Pluripotency capacity of the reporter hiPSCs was analysed by reverse trascription PCR and immunofluorescence.Three-germ-layer formation experiment was carried out to analyse the multi-lineage differentiation ability of the reporter hiPSCs.The reporter hiPSCs were further differentiated to obtain three-dimension (3D) retinal organoids, and immunofluorescence was used to identify the co-localization of the enhanced green fluorescent protein (eGFP) and VSX2.Results:A VSX2 eGFP reporter hiPSC clone was successfully obtained by CRISPR/Cas9 technology, which was consistent with the parental hiPSCs (BC1-hiPSCs) in morphology, without any chromosomal aberrations or cell line cross-contamination.Reverse transcription PCR assay and immunofluorescence showed obvious positive expressions of iPSCs markers in BC1- VSX2 eGFP-iPSCs, including NANOG, OCT4, SOX2, DNMT3B and GDF3 mRNA as well as NANOG, OCT4, SSEA4 and TRA-1-60 protein.The α-fetoprotein (AFP), α-smooth muscle actin (α-SMA) and neuronal class Ⅲ β-tubulin (TUJ1) were expressed in endoderm, mesoderm and ectoderm, respeetively, derived from BC1- VSX2 eGFP-iPSCs, and eGFP and VSX2 were co-stained in the neural retinal layer of 3D retinal organoids derived from BC1- VSX2 eGFP-iPSCs by immunofluorescence. Conclusions:VSX2 fluorescent reporter hiPSCs is successfully generated, which can monitor the temporal and spatial expression changes of VSX2 protein in real time, providing a powerful tool for evaluation of retina development mechanism and cell therapy.
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Objective:To evaluate the retinal differentiation ability of human induced pluripotent stem cells (hiPSCs) from various somatic cell sources.Methods:The hiPSCs lines BC1- green fluorescent protein (GFP) and Gibco obtained by blood cell reprogramming and the hiPSCs line UE017 obtained by urine cell reprogramming were used to induce retinal differentiation.The morphogenesis and development of retina were recorded with an optical microscope, and the expression of specific molecular markers of various cell subclasses in the retina was detected by immunofluorescence, and the efficiency of retinal differentiation of different cell lines was analyzed and compared.Results:All three hiPSC lines derived from blood and urine cells were able to be induced into three-dimensional (3D) retinal organoids, including neuroretina and retinal pigment epithelial cells.Retinal organoids simulated the development process of retina in vivo and gradually differentiated into all cell subtypes of retina, including retinal ganglion cells, photoreceptor cells, amacrine cells, horizontal cells, bipolar cells, Müller cells, and even formed lamellar structures.However, in terms of the efficiency of acquiring retinal organoids, the hiPSCs derived from blood were more efficient than those derived from urine. Conclusions:hiPSCs from both blood and urine somatic cells can differentiate into 3D retinal organoids, including all subtypes of retinal cells.The differentiation efficiency among lines is different.
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Purpose [WT5”BZ]To investigate the characteristics of intraocular growth of mice embryonic stem cells (ESC) in nude mice. [WT5”HZ]Methods [WT5”BZ]The undifferentiated murine ESC in vitro were transplanted into the eyes of nude mice.Mophological and immunohistochemical examinations were implemented. [WT5”HZ]Results [WT5”BZ]Two to three days after transplantation,yellowish white granules and masses were seen inside the anterior chamber and vitreous cavity and enlarged gradually.Morphological examination showed that there were undifferentiated cells and differentiated cells in anterior chamber and vitreous cavity.The morphology and alignment of some differentiated cells were similar to those of the retina of nude mice.The cells were highly positive in NSE staining. [WT5”HZ]Conclusion [WT5”BZ]The transplanted ESC could grow in the eyes of nude mice and differentiate into neurons and retina like structure. [WT5”HZ]
ABSTRACT
AIM: To investigate the intraocular growth characteristics of mice embryonic stem (ES) cells in nude mice.METHODS: Murine embryonic stem cells (D3 cell lines) were cultured and maintained in an undifferentiated state in vitro, then transplanted into the eyes of nude mice. In 6-45 d, the nude mice were executed for Morphological and immunohistochemical examinations.RESULTS: ES cells were developed into masses which enlarged gradually in the anterior chamber and vitreous cavity. Morphological examination showed different component: cysts, sheets and cords of medullary epithelium and rosettes in the eyes of the nude mice. Most of cells were highly stained by NSE, and some cells were moderately stained by GFAP.CONCLUSION: The embryonic stem cells(D3 cell lines) could differentiated into medulloepithelioma-like tissue in the anterior chamber and vitreous cavity of the Balb/c nude mice.
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AIM:To isolate and culture tumor stem cells in human retinoblastomas (RTSC). METHODS: Retinoblastoma (RB) single cells acquired from fresh tumors of RB patients by enzyme digestion were seeded in serum-free medium at a density of 1?10~8 cells/L. Clonal cultures were plated at a density of 1?10~6 cells/L. Secondary tumor spheres were triturated again and passaged in fresh medium. The sphere-forming, proliferation and differentiation assays were performed. RT-PCR and immunocytochemistry were performed to identify the RTSC and differentiated cells. RESULTS: All RB tumors studied produced proliferating neurosphere-like tumor spheres, which were also passaged multiple times. These tumor spheres had the capability to self-renew, proliferate in SFM medium, expressed retinal progenitor cell related genes, and differentiated into neurons and glia when they were transferred to differentiation conditions.CONCLUSION:Our findings demonstrated that there were subsets of tumor stem cells resembling retinal progenitor cells in human RB, which can be isolated, cultured in SFM. The RTSC may be original cells of RB tumor, and also become the new target of tumor therapy.