Biology subjects

Hoffer, D.

Publications and source records attributed to Hoffer, D..

3 recordsLinked to original sources

bioRxiv2024

Stimulating the regenerative capacity of the human retina with proneural transcription factors in 3D cultures

Retinal diseases often lead to degeneration of specific retinal cell types with currently limited therapeutic options to replace the lost neurons. Previous studies have reported that overexpression of ASCL1 or combinations of proneural factors in Muller glia (MG) induces regeneration of functional neurons in the adult mouse retina. Recently, we applied the same strategy in dissociated cultures of fetal human MG and although we stimulated neurogenesis from MG, our effect in 2D cultures was modest and our analysis of newborn neurons was limited. In this study, we aimed to improve our MG reprogramming strategy in a more intact retinal environment. For this purpose, we used an in vitro culture system of human fetal retinal tissue and adult human postmortem retina. To stimulate reprogramming, we used lentiviral vectors to deliver constructs with a glial-specific promoter (HES1) driving ASCL1 alone or in combination with additional developmental transcription factors such as ATOH1 and NEUROD1. Combining IHC, scRNA-seq and electrophysiology, we show for the first time that human MG can generate new neurons even in adults. This work constitutes a key step towards a future clinical application of this regenerative medicine approach for retinal degenerative disorders.

developmental biology↗

bioRxiv2024

Ciliary marginal zone of the developing human retina maintains retinal progenitor cells until late gestational stages

Non-mammalian vertebrates maintain a proliferative stem cell population at the far periphery of their retina called the ciliary marginal zone (CMZ), which gives rise to all retinal cell types and contributes to retinal regeneration upon injury. Humans do not maintain a proliferative CMZ into adulthood; however, it is not known how long in development this region continues to generate new neurons. Here, we identify a population of cells in the far peripheral retina of the fetal human that continues to proliferate long after the rest of the retina is quiescent. Single cell RNA-sequencing and EdU tracing at late time points in development reveal that this region has features of the non-mammalian CMZ, including the capacity to produce both early and late born cell types at late developmental stages, and a longer cell cycle than more centrally located retinal progenitor cells (RPCs). Moreover, while more central RPCs exit the cell cycle with the addition of a TGF{beta}-inhibitor, we show that early RPCs within the CMZ do not. These findings define the late stages of neurogenesis in human retinal development, and present a unique model system to study the fetal CMZ in humans.

developmental biology↗

bioRxiv2023

Single-cell sequencing of individual retinal organoids reveals determinants of cell fate heterogeneity

With a critical need for more complete in vitro models of human development and disease, organoids hold immense potential. Their complex cellular composition makes single-cell sequencing of great utility; however, the limitation of current technologies to a handful of treatment conditions restricts their use in screens or studies of organoid heterogeneity. Here, we apply sci-Plex, a single-cell combinatorial indexing (sci)-based RNA-seq multiplexing method to retinal organoids. We demonstrate that sci-Plex and 10x methods produce highly concordant cell class compositions and then expand sci-Plex to analyze the cell class composition of 410 organoids upon modulation of critical developmental pathways. Leveraging individual organoid data, we develop a method to measure organoid heterogeneity, and we identify that activation of Wnt signaling early in retinal organoid cultures increases retinal cell classes up to six weeks later. Our data show sci-Plexs potential to dramatically scale-up the analysis of treatment conditions on relevant human models.

genomics↗