Related Experiment Video
Updated: Jun 7, 2026
Isolation of Primary Murine Retinal Ganglion Cells (RGCs) by Flow Cytometry
Published on: July 5, 2017
[Study on the differentiation of retinal ganglion cells from rat Müller cells in vitro]
1Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China.
Objective:
To certify the ability of retinal Müller cells for producing neural stem cells in vitro and to find a method that can aquire more retinal ganglion cells from these stem cells.
Methods:
Müller cells were isolated from rat retina, and proliferating cells were expanded in serum-containing medium. The third or fourth passage of cells were identified by RT-PCR and Immunocytochemistry analysis.For dedifferentiation, the cultured cells were transferred to the sphere-culture medium composed of DMEM/F-12 supplemented with N2, bFGF and EGF. After 3 - 5 days, the culture media were substituted with BDNF, RA and 5% FBS and culture was continued for 7 - 10 days. At last, cells in this two stages were identified by immunocytochemical analysis.
Results:
Approximately (95.17 ± 2.68)% of cells in the culture were Müller cells as revealed by expressing glutamate-aspartate transporters (GLAST) and glutamine synthetase (GS) immunoreactivities. RT-PCR analysis also revealed that the culture was enriched for Müller cells and not contaminated with other retinal cells. After 3 - 5 days cultured in the the sphere-culture medium, the Müller cells became round and differentiate to neurospheres. (95.26 ± 1.35)% of cells in the neurosphere were positively reacted for Nestin, and (90.33 ± 4.12)% for BrdU. Neurospheres cultured for 7 - 10 days with 5% FBS, BDNF and RA can redifferentiate to various new cells. And the expression of Thy1.1 which is a marker of retinal ganglion cells was observed in (21.14 ± 1.49)% of these cells.
Conclusions:
Adult rodent Müller cells can generate clonal neurospheres, which consist of proliferating and multipotent cells, and redifferentiate to ganglion cells. This study may provide a novel tool in the study on stem cells and contribute to therapies for neural regeneration in retina.
Insights
Adult rodent Müller cells can generate neural stem cells in vitro. These cells can then be differentiated into retinal ganglion cells, offering a new approach for retinal regeneration therapies.
Area of Science:
- Cell Biology
- Neuroscience
- Stem Cell Research
Context:
- Retinal Müller cells, a type of glial cell in the retina, possess stem cell-like properties.
- Understanding Müller cell plasticity is crucial for developing regenerative therapies for retinal diseases.
Purpose:
- To investigate the potential of adult rodent Müller cells to dedifferentiate into neural stem cells in vitro.
- To establish a method for generating retinal ganglion cells from these derived stem cells.
Summary:
- Müller cells isolated from rat retina were cultured and induced to form neurospheres, demonstrating dedifferentiation into multipotent neural stem cells.
- These neurospheres were subsequently cultured under specific conditions to promote redifferentiation, yielding cells expressing markers of retinal ganglion cells, including Thy1.1.
Impact:
- This research provides a novel method for generating retinal ganglion cells from Müller cells, potentially advancing stem cell-based therapies for retinal regeneration.
- The findings offer a new tool for studying stem cell biology and developing treatments for vision loss due to retinal damage or degeneration.
