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Application of New Human ESC Reporter Cell Line Enhances Differentiation of Important Retinal Cells



Review of “Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells” from STEM CELLS Translational Medicine by Stuart P. Atkinson

Retinal ganglion cell (RGC) axons serve as a connection between the retina and the brain and any damage or disease that affects these cells can lead to vision loss. Cell replacement with RGCs derived from pluripotent stem cells (PSCs) represents an exciting therapeutic strategy that the labs of Donald J. Zack (Johns Hopkins University School of Medicine, Maryland, USA) and Derek S. Welsbie (University of California San Diego, USA) have actively followed.

The lab´s previous study detailed the generation of an RGC-specific reporter human embryonic stem cell (hESC) line to assess differentiation protocols via fluorescence-activated cell sorting (FACS) [1]. However, the team has now returned with an improved reporter hESC line that combines fluorescence with immunopurification to permit the evolution of a differentiation protocol that promises the facile, efficient, and scalable production of human RGCs for clinical applications [2].

Sluch et al. first employed CRISPR/Cas9 genome-editing technology to engineer hESCs expressing a cassette coding for a fluorescent marker, to allow detection, and the murine THY1.2 cell surface protein, to allow for RGC-specific cell immunopurification, under the control of an RGC-specific gene promoter (BRN3B). Employing their previously described RGC differentiation protocol [3] combined with magnetic microbead-based immunopurification, the authors achieved an RGC yield of around 70%. However, the application of this newly engineered hESC line permitted the further optimization and simplification of the differentiation protocol via the modulation of RGC-associated signaling pathways. With the possible clinical translation of RGCs in mind, these steps will reduce costs, increase yields, and therefore enhance clinical applicability. Encouragingly, resultant RGCs highly resembled endogenous RGCs, as evidenced by RNA-sequencing and immunocytochemical comparisons, and the authors demonstrated their utility as part of an in vitro axonal injury model suitable for the discovery of essential RGC pathways (e.g., DLK signaling [4]) and a high-throughput screening for neuroprotective compounds (e.g., tozasertib [4]).

Given the potential for large-scale production following the optimization and simplification steps afforded by this newly engineered reporter hESC line, the authors posit that RGC-based high-throughput drug discovery and/or biochemical inquiries will soon follow. Furthermore, this study proposes the application of CRISPR/Cas9 genome editing and immunopurification as an exciting means to enhance the differentiation human PSCs into any cell type for use in drug screens, disease modeling, and perhaps even cell replacement therapies.

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  1. Sluch VM, Davis CH, Ranganathan V, et al., Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line. Sci Rep 2015;5:16595.
  2. Sluch VM, Chamling X, Liu MM, et al., Enhanced Stem Cell Differentiation and Immunopurification of Genome Engineered Human Retinal Ganglion Cells. STEM CELLS Translational Medicine 2017;6:1972-1986.
  3. Crombie DE, Daniszewski M, Liang HH, et al., Development of a Modular Automated System for Maintenance and Differentiation of Adherent Human Pluripotent Stem Cells. SLAS Discov 2017;22:1016-1025.
  4. Welsbie DS, Yang Z, Ge Y, et al., Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death. Proc Natl Acad Sci U S A 2013;110:4045-50.