Abstract
Reporter lines generated in human pluripotent stem cells can be highly useful for the analysis of specific cell types and lineages in live cultures. We created the first human rod reporter line using CRISPR/Cas9 genome editing to replace one allele of the Neural Retina Leucine zipper (NRL) gene with an eGFP transgene in the WA09 human embryonic stem cell (hESC) line. After confirming successful targeting, three-dimensional optic vesicle structures were produced to examine reporter specificity and to track rod differentiation in culture. The NRL+/eGFP hESC line robustly and exclusively labeled the entirety of rods throughout differentiation, eventually revealing highly mature structural features. This line provides a valuable tool for studying human rod development and disease and testing therapeutic strategies for retinitis pigmentosa.
Highlights
Human pluripotent stem cell reporter lines permit identification of cell lineages and subtypes with visualization of morphology in live cultures
Using a modified version of our previously described optic vesicle (OV) differentiation protocol, we demonstrate production of a high percentage of rods from the Neural Retina Leucine zipper (NRL)+/eGFP human embryonic stem cell (hESC) line that are capable of self-assembling within an organized outer nuclear layer and assuming a mature rod structure replete with developing outer segments, as has been shown previously in unmodified Human pluripotent stem cell (hPSC) lines[24,25,26,27,28]
Genotyping was performed with primers that distinguished the unedited NRL allele from successfully are shown at day (d) 6. (B) embryoid bodies (EBs) are plated, whereupon they form OV-like structures (d26 shown). (C) After dissection, OVs are maintained long-term in suspension cultures (d30 shown). (D–G) At d30, NRL+/eGFP OVs consist mainly of an outer layer of VSX2+ (D) and Ki67+ (E) proliferative retinal progenitor cells (merged image in (F), and an inner layer of SNCG+ retinal ganglion cells (G)
Summary
Human pluripotent stem cell (hPSC) reporter lines permit identification of cell lineages and subtypes with visualization of morphology in live cultures. From a photoreceptor (PR) standpoint, all hPSC reporter lines generated far utilized the cone rod homeobox (CRX) gene to drive transgene expression[8,9] These CRX-based hPSC reporters robustly mark all PRs from early to late differentiation. They cannot distinguish rods from cones[8,9], nor are they inherently PR-specific since CRX is known to be expressed in other human retinal cell types[10]. The availability of a NRL hPSC reporter line that faithfully and innocuously labels human rods throughout differentiation should prove useful for numerous applications of stem cell technology, those related to the study and treatment of retinitis pigmentosa
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