Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells

Abstract

Human pluripotent stem cells have the potential to promote biological studies and accelerate drug discovery efforts by making possible direct experimentation on a variety of human cell types of interest. However, stem cell cultures are generally heterogeneous and efficient differentiation and purification protocols are often lacking. Here, we describe the generation of clustered regularlyinterspaced short palindromic repeats(CRISPR)-Cas9 engineered reporter knock-in embryonic stem cell lines in which tdTomato and a unique cell-surface protein, THY1.2, are expressed under the control of the retinal ganglion cell (RGC)-enriched gene BRN3B. Using these reporter cell lines, we greatly improved adherent stem cell differentiation to the RGC lineage by optimizing a novel combination of small molecules and established an anti-THY1.2-based protocol that allows for largescale RGC immunopurification. RNA-sequencing confirmed the similarity of the stem cell-derived RGCs to their endogenous human counterparts. Additionally, we developed an in vitro axonal injury model suitable for studying signaling pathways and mechanisms of human RGC cell death and for high-throughput screening for neuroprotective compounds. Using this system in combination with RNAi-based knockdown, we show that knockdown of dual leucine kinase (DLK) promotes survival of human RGCs, expanding to the human system prior reports that DLK inhibition is neuroprotective for murine RGCs. These improvements will facilitate the development and use of largescale experimental paradigms that require numbers of pure RGCs that were not previously obtainable. STEM CELLS TRANSLATIONAL MEDICINE 2017;6:1972-1986 SIGNIFICANCE STATEMENTPluripotent stem cells provide access to a large variety of human cell types. However, stem cell culture is often heterogeneous and efficient differentiation and purification of stem cell-derived cells are limited to relatively few examples. Via genetic engineering, we have generated stem cell reporter lines that enable the detection and purification of retinal ganglion cells (RGCs), an essential cell type for vision. Through modulation of known signaling pathways, we report an improved RGC differentiation protocol for high yields of purified RGCs. We also describe an siRNA protocol for exploration of signaling pathways in human RGCs.

Dual SMAD inhibition and Wnt inhibition enable efficient and reproducible differentiations of induced pluripotent stem cells into retinal ganglion cells

“…[28] Generation of a CRISPR engineered iPSC line with a fluorescent RFP tag reporter in the Brn3B locus, greatly assisted in evaluation of pathways necessary for RGC differentiation and characterization. [59] We developed a quick and efficient RGC generation protocol involve embryoid bodies or need retinal rosette production or enrichment to initiate RGC differentiation. In our method, the entire pluripotent iPSC culture is differentiated into RPCs in 21 days using a chemically defined medium.…”

“…This is a must faster timeline considering other published methods to date. [24,28,[59][60][61]. We used the a modified chemically defined media composition standardized by Teotia et al, 2017 [28] to take advantage of 15-day RGC differentiation and paired it with our novel RPC generation protocol to generate RGCs making our method unique.…”

Astrocytes Regulate the Development and Maturation of Retinal Ganglion Cells Derived from Human Pluripotent Stem Cells

“…Additionally, the definitive identification of RGCs within such cultures has been complicated by the lack of unique, reliable markers that allow for the visualization of entire RGCs including their extensive neurites. Thus, to overcome these issues, a CRISPR/Cas9-engineered cell line with a tdTomato reporter and mouse Thy1.2 selectable marker was used to identify and enrich for hPSC-derived RGCs (Sluch et al, 2017).…”

Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types

“…CRISPR/Cas9 gene editing greatly expands the ability to insert cell-type-specific reporter genes into hiPSCs for this purpose. Although several hiPSCs with single reporter knock-ins exist [5][6][7] , these lines only detect terminally differentiated retinal cell types. This limits the ability to study progression of retinal development in real time.…”

“…Furthermore, we functionally characterized a single, multi-targeted hiPSC clone (PGP1) both by appropriate fluorescent protein expression and by differentiation into all major retinal cell types during retinal organoid formation. Although previous publications have described single retinal cell-type-specific reporters, to our knowledge, PGP1 represents the first triple targeted retinal reporter hiPSC clone[5][6][7] . The ability of the PGP1 line to facilitate visual observation of retinal cell differentiation, without compromising retinal organoid formation typical of WT hiPSCs16 , makes this clone particularly useful for a number of different applications.The PGP1 line provides a powerful tool for real time retinal disease modeling.…”