Using Zinc Finger Nuclease Technology to Generate CRX-Reporter Human Embryonic Stem Cells as a Tool to Identify and Study the Emergence of Photoreceptors Precursors During Pluripotent Stem Cell Differentiation

Abstract

The purpose of this study was to generate human embryonic stem cell (hESC) lines harboring the green fluorescent protein (GFP) reporter at the endogenous loci of the Cone‐Rod Homeobox (CRX) gene, a key transcription factor in retinal development. Zinc finger nucleases (ZFNs) designed to cleave in the 3′ UTR of CRX were transfected into hESCs along with a donor construct containing homology to the target region, eGFP reporter, and a puromycin selection cassette. Following selection, polymerase chain reaction (PCR) and sequencing analysis of antibiotic resistant clones indicated targeted integration of the reporter cassette at the 3′ of the CRX gene, generating a CRX‐GFP fusion. Further analysis of a clone exhibiting homozygote integration of the GFP reporter was conducted suggesting genomic stability was preserved and no other copies of the targeting cassette were inserted elsewhere within the genome. This clone was selected for differentiation towards the retinal lineage. Immunocytochemistry of sections obtained from embryoid bodies and quantitative reverse transcriptase PCR of GFP positive and negative subpopulations purified by fluorescence activated cell sorting during the differentiation indicated a significant correlation between GFP and endogenous CRX expression. Furthermore, GFP expression was found in photoreceptor precursors emerging during hESC differentiation, but not in the retinal pigmented epithelium, retinal ganglion cells, or neurons of the developing inner nuclear layer. Together our data demonstrate the successful application of ZFN technology to generate CRX‐GFP labeled hESC lines, which can be used to study and isolate photoreceptor precursors during hESC differentiation. Stem Cells 2016;34:311–321

A Novel Approach to Single Cell RNA-Sequence Analysis Facilitates In Silico Gene Reporting of Human Pluripotent Stem Cell-Derived Retinal Cell Types

“…This is important, since even targeting a reporter to an endogenous locus can fail to recapitulate the full expression profile of a particular gene. Indeed, our CRX +/tdTomato hPSC line, as well as the allele‐targeted CRX reporter line generated by the Lako laboratory , did not label BPCs, a known CRX+ NR cell type. This omission may be due to disruption of cryptic BPC‐specific regulatory sequences at or distal to the reporter gene insertion site.…”

“…Methods to assess the composition of complex cultures are numerous and include gene reporters and single‐cell analytical techniques. To date, hPSC reporter lines have been helpful in broadly evaluating NR progenitor cells (NRPCs) and photoreceptors (PRs) . In particular, comparative bulk RNA‐sequence (seq) analysis on sorted retinal cells has provided insight into the pooled transcriptomes of all PRs present within early organoids .…”

Systematic Comparison of Retinal Organoid Differentiation from Human Pluripotent Stem Cells Reveals Stage Specific, Cell Line, and Methodological Differences

“…The production of photoreceptors from human stem cells was originally reported under two‐dimensional (2D) culture conditions which gave rise to photoreceptor precursor cells and some photoreceptors expressing mature photoreceptor markers . This was then achieved in three‐dimensional (3D) culture, which gave rise to developing photoreceptors within optic vesicle‐like structures that consisted of multiple retinal progeny within distinct retinal laminae . This advance enabled the production of retinal tissue, which more closely mimicked the natural order of retinogenesis and microarchitecture of the native retina than 2D approaches had allowed.…”

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.…”