Function of human pluripotent stem cell-derived photoreceptor progenitors in blind mice

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions, resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration, these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals, as evidenced by two visual behavioral tests. Furthermore, the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease.


Figure S11. Control measures for functional improvement of vision
In order to control for differences in behavior of the three treatment group in the light avoidance assay, transitions between the arena chambers and the distance travelled by animals within the lit chamber were also assessed during the experiments as behavioral measures of anxiety in rd1 mice. There were no differences between the three groups in (a) mean number of transitions between the light and dark chambers (F=0.9014, p=0.4211 [ns]) or (b) the mean distance (meters) traveled within the lit chamber (F=0.1297, p=0.8790, ns) throughout the test. Dashed line represents the mean response of age-matched wild-type mice. One way ANOVA, n=8 per group, ns, non-significant.

Figure S12. Control measures for morphology and number of residual host cones
To account for the possibility of improvement in visual function resulting from host cone neuroprotection by transplanted cells, the number of residual cones was assessed by cone arrestin staining. Image a. shows cone arrestin staining of an adult WT retina, with cone bodies, processes and inner and outer segment stained (red). b Residual cones (red) in the adult rd1 mouse three weeks after transplantation of ESC-PhRP (green). Host cone cells show abnormal morphology and absence of inner and outer segments. c. Residual host cones were quantified three weeks following transplantation, and no difference was found in the number of residual host cones between mice treated with ESC-PhRP, iPSC-PhRP or sham injection (F=0.56, p=0.58, ns; One way ANOVA, n=5 specimens per group). Error bars represent ±1 S.E.M. INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelial; ns, non-significant.

Culture of undifferentiated human PSCs
Human control. Data was analyzed using BD Accuri C6 software.

AAV vector generation
The Y444F mutation was created in the Rep2Cap2 plasmid using the QuikChange II site-directed mutagenesis kit (Agilent Technologies) as per instructions with the HPLC purified forward primer: CAGTACCTGTATTTCTTGAGCAGAACAAACAC and reverse primer: TTTGTTCTGCTCAAGAAATACAGGTACTGGT. The transgene plasmid (pTransgene) was generated by isolating the 199bp human rhodopsin kinase promoter (GRK1) fragment previously described [2]. This was attached to the exon/intron elements of the CAG promoter using the SPLICE technique [3]. In

Cell cryopreservation
For long term storage and cell shipment, PhRP spheres were cryopreserved in an-animal-free cell cryopreservation buffer, Cryostor CS10 (BioLife Solutions, Inc). 5 million cells were frozen in a final volume of 1 ml of Cryostor CS10 medium. Then stored at -80C for 2 days, and transferred to liquid nitrogen. Frozen cells were rapidly thawed in a 37°C water bath and cells viability was assayed by Trypan Blue (sigma) exclusion.
We routinely check cell viability after cell cryopreservation. Viability was tested immediately post thawing, again at 48 hours and finally prior to transplantation. Using the described method, we typically obtained 70-85% viable cells post thawing. Only batches with over 75% viable cells were used for further application. Following media replacement and removal of dead cells, we tested viability again at 48 hours and immediately prior to transplantation. Over 90% cell viability was measured in cohorts at 48 hours and at the day of transplantation. were housed together to reduce variability between treatment groups.

Cell transplantation
Transplantations were performed by subretinal injection under direct visualization using an operating microscope (M620 F20, Leica, Wetzlar, Germany). The pupils of 10-12 week old mice were dilated as described above and a liquid gel (Viscotears, Novartis, Frimley, UK) was applied to the eye. A 6mm circular cover glass was positioned over the cornea to allow visualization of the retina. Cell suspensions were transplanted subretinally using a Hamilton syringe and a 10mm 34-gauge needle (65N, Hamilton AG) inserted into the subretinal space though the sclera as previously described [5]. 2 µl of diluted cells (Approximately 2 × 10 5 cells were transplanted in each injection) or buffer (PBS) was delivered unilaterally into the subretinal space of the right eye.

Scanning Laser Ophthalmoscopy
Autofluorescence (AF) imaging was performed 3 weeks post transplantation using a confocal scanning laser ophthalmoscope (cSLO; Spectralis HRA, Heidelberg Engineering, Heidelberg, Germany) as previously described [6]. Animals were anaesthetised and pupils were dilated as above. for immunohistochemistry and further analysis.

In vitro immunocytochemistry
Cells were fixed in 4% paraformaldehyde for 30 min at room temperature.
Samples were blocked with 5% normal goat or donkey serum (Jackson ImmunoResearch) and 0.3% Triton X-100 in PBS at room temperature for 1 h, followed by incubation with primary antibodies at room temperature for 1 h. There

Light microscopy
Quantitative analysis of GFP expression in transduced neural spheres and retinal sections was achieved by light microscopy imaging, using Leica DM IL inverted epifluorescence microscope. Images were obtained using identical acquisition setting and exposure time for comparable slides and were saved at a resolution of 1200x1600 pixels.

Confocal Microscopy
Retinal sections were viewed on a confocal microscope (LSM710; Zeiss, Jena, Germany). The fluorescence of Hoechst, GFP, Alexa-555 and Alexa-635 was excited using 350-nm UV, 488-nm argon, and the 543-nm HeNe lasers, as appropriate. GFP-positive cells were first located using epifluorescence illumination and then a series of XY optical sections (approximately 0.5µm thickness) were taken in succeeding stacks to give XY projection images. Image processing was performed using Image J (Version 1.47, National Institute of Health, http://rsb.info.nih.gov/ij/index.html).

Immune suppression
Animals were immune suppressed by addition of cyclosporine A (50 mg/kg/day) and 5% fruit cordial to the drinking water [7] for 2 days prior to and 3 weeks following transplantation.

Statistical analysis
Cell quantification was analyzed using two-tailed Student t tests.
Optomotor response data was analysed using a paired student t test or one-way