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

Autor: Donald J. Zack, Jie Cheng, Xitiz Chamling, Derek S. Welsbie, Katherine L. Mitchell, Melissa M. Liu, Cynthia A. Berlinicke, Valentin M. Sluch
Rok vydání: 2017
Předmět:
Pluripotent Stem Cells
Retinal Ganglion Cells
0301 basic medicine
Cellular differentiation
Human Embryonic Stem Cells
Stem cells
Biology
Stem cell marker
Retinal ganglion
Cell Line
03 medical and health sciences
Translational Research Articles and Reviews
medicine
Humans
Gene Delivery Systems / Gene Therapy / Gene Editing Technology
Cellular Reprogramming Techniques
Clustered regularly interspaced short palindromic repeats
Induced pluripotent stem cell
Embryonic Stem Cells
Cells
Cultured
Gene Editing
Developmental Biology / Embryo Development
Cell Differentiation
Cell Biology
General Medicine
MAP Kinase Kinase Kinases
Embryonic stem cell
Molecular biology
Transcription Factor Brn-3B
Neural/Progenitor Stem Cells
3. Good health
Cell biology
030104 developmental biology
medicine.anatomical_structure
Retinal ganglion cell
Disease Models (Animal/Cell)
Cell culture
Vision Loss / Repair
sense organs
CRISPR-Cas Systems
Stem cell
Biotechnology
Developmental Biology
Zdroj: Stem Cells Translational Medicine
ISSN: 2157-6580
2157-6564
Popis: 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 regularly-interspaced 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 large-scale 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 large-scale experimental paradigms that require numbers of pure RGCs that were not previously obtainable.
Databáze: OpenAIRE
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