Abstract
Purpose :
In mammals, loss of retinal cells due to disease or trauma is an irreversible process which leads to blindness. Interestingly, regeneration of retinal neurons is a well-established process in some non-mammalian vertebrates and is driven by the Muller glia (MG) which are able to re-enter the cell cycle and reprogram into neurogenic progenitors upon retinal injury or disease. Progress has been made to “translate” this mechanism to mammals to promote retinal regeneration. A breakthrough in the field was the demonstration that MG can generate new neurons in vivo in the adult mouse retina after the overexpression of the pro-neuronal transcription factor Ascl1. In this study, we show that the same strategy could be applied to regenerate retinal neurogenic progenitors using human MG in vitro.
Methods :
In human fetal development, MG appear following a temporal central to periphery gradient in the retina and, can first be identified at Day 73 in the center and later (D100) in the periphery. We have developed protocols for obtaining in vitro 2D cultures of MG dissociated from stem cell-derived organoids over 150 days of age. MG cultures were characterized with immunostaining and single cell RNA sequencing (scRNA-seq). We used lentivirus mediated gene delivery for over-expression of ASCL1 (CMV-ASCL1-GFP). The addition of EdU to the culture combined with GFP allowed us to track newly generated cells to control for any surviving neurons from the initial dissociation of the organoids. To determine whether ASCL1 induces neurogenesis in human MG, we used scRNA-seq analysis and Immunolabeling (IHC) for neuro-specific antibodies and EdU.
Results :
IHC analysis showed that MG are present in retinal organoids around Day 180. MG derived from organoids can be grown and passaged in 2D cultures, are of good purity and contain only few surviving neurons. MG can also be frozen for cell banking. When infected with a lentivirus driving ASCL1-GFP, the human MG can be reprogrammed to a neurogenic state as assessed with IHC, and by using scRNA-seq. Our study demonstrates for the first time that Ascl1 can reprogram human MG into neurogenic progenitor cells in vitro.
Conclusions :
Our work demonstrates the first evidence of regenerative capacity of MG in human.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.