Abstract
Purpose :
The aim of this study is to clarify the developmental characteristics and transcriptome profile of Müller glial cells (MGCs) in an human induced pluripotent stem cells (hiPSCs)-derived retinal organoids (ROs), and establish protocols to enrich these cells, thus to facilitate the basic and translational study of MGC-related retinal diseases.
Methods :
hiPSCs were maintained with mTeSR1, and differentiated into ROs according to our published protocol. ROs were sequentially collected to evaluate the temporal and spatial development of MGCs in ROs . Neural retina (NR) of ROs were collected and dissociated into single cells to amplify MGCs under adherent culture. Optical microscope was used to observe the morphology of the passaged MGCs, RNA-Seq and immunofluorescence staining to detect the expression of specific MGCs markers, and electronic microscopy to observe the ultrastructure of MGCs. The functional features of MGCs were also evaluated by the whole cell membrane clamp.
Results :
In ROs, retinal progenitor cells (RPCs) progressively differentiated into SOX9+ Ki67- MGC precursors during differentiation day (D) 60 to D90, while mature MGCs markers CRALBP and GS did not appear until D120. Specific transcriptome profiles of MGCs were revealed by RNA-seq with ROs. Cells isolated from ROs aged older than D120 was an optimal source for the enrichment of iMGCs with high purity and expansion ability. They had typical features of human MGCs in morphological, molecular and functional aspects, and could be passaged serially at least 10 times, yielding large number of cells in a short period. The transcriptome pattern of the expanded MGCs was also revealed. MGC-related genes such as VIM, CRYAB, CD44, DBI and CCL2 were enriched in the passaged MGCs, while other retinal cell markers were down-regulated. SEM and TEM showed that MGCs had many microvillus projections, large nucleus with obvious nucleoli. The whole cell membrane clamp showed MGCs could depolarize in response to L-glutamate puff.
Conclusions :
This study firstly clarified the timecourse of human MGC development in the RO model, where the MGCs could be enriched and expanded. The transcriptome pattern of MGCs was also revealed. These findings will pave the way for downstream investigation and application of MGC related retinal disorders.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.