Non-invasive retinal imaging of fluorescent hESC-derived photoreceptor precursors in the living primate

Abstract

Purpose : The efficacy of stem cell injections into the retina to treat vision loss is conventionally evaluated post-mortem using histological methods in animal models. We explore the use of optical coherence tomography (OCT) and high resolution adaptive optics scanning light ophthalmoscopy (AOSLO) as non-invasive imaging methods to longitudinally monitor the spatial distribution, longevity and structural remodeling of photoreceptor precursor cells in the living macaque. To allow the specific identification of transplanted photoreceptor precursors in vivo we created a human embryonic stem cell (hESC)-derived CRX reporter line expressing the fluorescent protein tdTomato.

Methods : Gene targeting via homologous recombination was used to create the hESC CRX:tdTomato line, and 3D optic vesicle-like structures were generated using previously published protocols established by the Gamm lab. Photoreceptor precursor cells were dissociated and suspended in HBSS before subretinal injection into a 15-year-old male macaque (Macaca fascicularis) using a 41-gauge needle and a 20 psi pressure pulse. Immune suppression was provided by daily subcutaneous injection of cyclosporine (17.5 mg). Reflectance (796nm) and fluorescence (excitation 561 nm, emission 584-676 nm) AOSLO images were collected over a period of three months post-transplant. OCT (Heidelberg Spectralis) was conducted in regions expressing Tdtomato.

Results : Photoreceptor precursors expressed tdTomato for more than three months post transplantation, implying cell survival during that period. Precursor cell aggregates continued to remodel during that time with OCT data indicating a two-fold increase in aggregate thickness between day 52 and day 114 post-transplant. AOSLO imaging showed ring-like groupings of cells with median diameter 95 µm, similar to ‘rosettes’ previously seen in histological studies. Locations associated with photoreceptor precursors were hyper-reflective.

Conclusions : Survival, distribution and remodelling of fluorescently-labelled photoreceptor precursors injected into the subretinal space can be tracked non-invasively using AOSLO and OCT. These non-invasive methods can be used for long-term in vivo monitoring of stem cell transplants at a cellular spatial scale, reducing the need to sacrifice animals at a range of time points. This approach could be especially valuable applied to human therapy.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.