研究目的
Evaluating the effects of a novel biomimetic polybenzyl glutamate (PBG) scaffold on culturing iPSC-derived RGC progenitors to promote differentiation and neurite outgrowth.
研究成果
The PBG scaffold promotes hiPSC differentiation toward retinal lineage, forms organoid-like structures, enhances neurite outgrowth, and shortens the differentiation process. It may be related to glutamate signaling pathways and has potential for disease modeling, pharmacological screening, and clinical applications.
研究不足
Aggregated retinal progenitors were used, making it difficult to harvest pure RGC progenitors for analysis, potentially underestimating statistical differences. The study did not address safety of transplantation or optimal timing for clinical application.
1:Experimental Design and Method Selection:
The study used a modified SFEB method for hiPSC differentiation to RGC-like cells, involving culture on PBG scaffold or cover glass controls, with RNA-seq and WGCNA for analysis.
2:Sample Selection and Data Sources:
Human-induced pluripotent stem cells (hiPSCs) from Gibco? Human Episomal iPSC Line were used, cultured on feeder layers and differentiated. RNA-seq data compared with undifferentiated iPSCs and mature retinal tissues from GEO datasets.
3:List of Experimental Equipment and Materials:
Equipment includes phase contrast microscope, SEM (JEOL JSM-6700F), real-time PCR system (Applied Biosystems 7500 Fast), electrospinning apparatus (syringe pump, power supply, metal collector), and various chemicals and antibodies. Materials include PBG scaffold, Matrigel, BDNF, antibodies for immunofluorescence.
4:Experimental Procedures and Operational Workflow:
hiPSCs were dissociated, reaggregated in RDM, transferred to RMM, and adherently cultured on PBG scaffold or cover glass with BDNF. Cells were harvested for mRNA expression, immunofluorescence, SEM observation, and RNA-seq analysis at specific time points.
5:Data Analysis Methods:
Real-time PCR with ΔΔCT method, ImageJ for neurite measurement, RNA-seq data analyzed with ExAtlas and WGCNA, statistical analysis using Mann-Whitney U test, t-test, and ANOVA.
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