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Posts Tagged ‘Human embryonic stem cells’

Photo-Receptor Production

Posted in Auditory and vision, Curation, Neuroscience, Regenerative Biology and Medicine, Signaling, Stem Cells for Regenerative Medicine, tagged , , , on March 15, 2016| Leave a Comment »

Photo-Receptor Production

Curator: Larry H. Bernstein, MD, FCAP

 

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

Joseph Collin1, Carla B Mellough1, Birthe Dorgau1, Stefan Przyborski2, Inmaculada Moreno-Gimeno3 and Majlinda Lako1,*

STEM CELLS Feb 2016  34(2), pages 311–321,    http://dx.doi.org:/10.1002/stem.2240

 

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 New Tool for Photoreceptor Production to Treat Vision Loss

     

Review of “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” from Stem Cells by Stuart P. Atkinson

The production of replacement cells from human pluripotent stem cell (hPSC) sources has great potential for the treatment of certain forms of vision impairment and blindness. The production of functional stem cell-derived retinal-pigmented epithelium (RPE) is already a notable success, although the equivalent success in photoreceptor cell production has so far lagged behind, due partly to the lack of robust human cell surface markers to allow their purification.

To get round this problem, canny researchers from the laboratory of Majlinda Lako (Newcastle University, United Kingdom) have used zinc finger nuclease (ZFN) gene editing technology to create a reporter embryonic stem cell (ESC) line suitable for the enhanced production of photoreceptor cells [1].

The authors targeted a green fluorescent protein (GFP) reporter into the endogenous locus of the Cone-Rod Homeobox (CRX) transcription factor gene which is known to be selectively expressed post-mitotic retinal photoreceptor precursors. The integration of this reporter into hESCs did not negatively affect genomic stability or pluripotency and, following 3D differentiation to form laminated neural retina [2], GFP expression faithfully mimicked the known expression patterns of CRX (See Figure).

In-depth expression analysis of CRX-positive cells then demonstrated the restriction of GFP-CRX to only two cell types within the 90-day differentiation protocol: RECOVERIN-expressing photoreceptor precursors situated in the developing outer nuclear layer of the optic cup and a subpopulation of non-proliferative retinal progenitors. Importantly, the study detected the expression of genes known to be activated by CRX, so suggesting that GFP-targeting does not affect the functionality of the transcription factor.

In conclusion, the authors have created a CRX-GFP-labeled hESC line which can be used to identify, purify, and study photoreceptor precursors during hESC differentiation, in the hope of improving differentiation protocols, discovering cell surface markers, and developing clinically applicable strategies for transplantation. A great tool for those working towards generating treatments for vision impairment and blindness.

References

  1. Collin J, Mellough CB, Dorgau B, et al. 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. STEM CELLS 2016;34:311-321.
  2. Mellough CB, Collin J, Khazim M, et al. IGF-1 Signaling Plays an Important Role in the Formation of Three-Dimensional Laminated Neural Retina and Other Ocular Structures From Human Embryonic Stem Cells. Stem Cells 2015;33:2416-2430.

 

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