Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-Derived Retinal Organoids and Fetal Retinae

Abstract

Loss of photoreceptor cells due to retinal degeneration is one of the main causes of blindness in the developed world. Although there is currently no effective treatment, cell replacement therapy using stem‐cell‐derived photoreceptor cells may be a feasible future treatment option. In order to ensure safety and efficacy of this approach, robust cell isolation and purification protocols must be developed. To this end, we previously developed a biomarker panel for the isolation of mouse photoreceptor precursors from the developing mouse retina and mouse embryonic stem cell cultures. In the current study we applied this approach to the human pluripotent stem cell (hPSC) system, and identified novel biomarker combinations that can be leveraged for the isolation of human photoreceptors. Human retinal samples and hPSC‐derived retinal organoid cultures were screened against 242 human monoclonal antibodies using a high through‐put flow cytometry approach. We identified 46 biomarkers with significant expression levels in the human retina and hPSC differentiation cultures. Human retinal cell samples, either from fetal tissue or derived from embryonic and induced pluripotent stem cell cultures, were fluorescence‐activated cell sorted (FACS) using selected candidate biomarkers that showed expression in discrete cell populations. Enrichment for photoreceptors and exclusion of mitotically active cells was demonstrated by immunocytochemical analysis with photoreceptor‐specific antibodies and Ki‐67. We established a biomarker combination, which enables the robust purification of viable human photoreceptors from both human retinae and hPSC‐derived organoid cultures. Stem Cells 2018;36:709–722

Generation of human neural retina transcriptome atlas by single cell RNA sequencing

“…The establishment of reference transcriptome maps for individual cell types in the retina provide unprecedented insights into the signals that define retinal cell identity and advance our understanding of the retina. This human neural retina transcriptome data can be used as a benchmark to assess the quality and maturity of pluripotent stem cell-derived retinal cells, such as retinal ganglion cells (Gill et al, 2016;Kobayashi et al, 2018;Sluch et al, 2015) and photoreceptors (Lakowski et al, 2018). We also confirmed that a relatively low level of sequencing depth (~40,232 reads per cell) is sufficient for identification and classification of major cell types in a complex tissue like retina.…”

CRX Expression in Pluripotent Stem Cell-Derived Photoreceptors Marks a Transplantable Subpopulation of Early Cones

“…For cell based therapies, it is important to generate and enrich defined populations of retinal cells of interest (e.g., photoreceptors, RPE cells) and assess their transplantation into the context of host retinal environment. Various approaches have been used to selectively enrich retinal cell types from these complex organoids including immunostaining with cell surface markers or flow activated cell sorting using reporter labeled cell lines , which harbor fluorescent markers of key photoreceptor transcription factors such as cone rod homeobox ( CRX ) or neural retina leucine zipper ( NRL ) genes. Enrichment of photoreceptors is, however, not sufficient for ensuring successful engraftment into an adult retina.…”

Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent

“…Induced pluripotent stem cell (IPSC)-derived retinal organoids have been shown to have a wide range of applications, including the study of human retinogenesis, 1-3 disease modeling, 4,5 drug discovery, 6,7 and cell therapy. [8][9][10] Numerous protocols have been developed for the generation of retinal organoids that follow basic developmental principles of forebrain development and eye formation. Despite the ability of these protocols to give rise to laminated retinal organoids, variability in the propensity of iPSCs to give rise to various retinal cell types is often reported.…”