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Variation in the Phenotype of Photosensitive Cells Produced from Human Fibroblast Cell Lines.

BACKGROUND
Photoreceptors differentiated from somatic cells are a useful tool for transplantation and drug screening. We previously showed that photosensitive cells are differentiated from human fibroblasts by direct reprogramming. In induced pluripotent stem (iPS) cells or embryonic stem (ES) cells, the properties of differentiated cells differ among the source of cell lines. However, whether or not the properties of the photosensitive cells produced by direct reprogramming are controlled by the origin of the cell line remains unknown.


METHODS
We compared the morphological and physiological properties of photosensitive cells induced by two fibroblast cell lines.


RESULTS
The differentiated cells had larger somas and more primary processes than the non-infected cells in both cell lines. The degree of morphological change was statistically different between the two cell lines. In addition, physiological responses to light differed between the two cell lines. An outward current (photoreceptor-like response) was observed in both cell lines, while an inward current (intrinsically photosensitive retinal ganglion cell-like response) was observed only in one cell line under light stimulation.


CONCLUSIONS
These results suggest that photosensitive cells produced from different cell lines by direct reprogramming might express different phenotypes.


Introduction
Retinitis pigmentosa, age-related macular degeneration, and cone dystrophy lead to a loss of photoreceptors and retinal pigment epithelium. One feasible therapy for retinal disease is regenerative medicine. Photoreceptors derived from embryonic stem (ES) cells 1  Increasing evidence has recently shown that the type of ES and iPS cell lines affect the ability of cells to differentiate into specific cells 6 9 . In addition, the phenotypes of iPS cell lines differ 6 . These findings support the notion that variations of the intrinsic properties among original cells are represented by the phenotypes of the differentiated cells produced from iPS cell lines. Therefore, how much phenotypic variation is represented among cell lines, considering that photosensitive cells can be produced by direct reprogramming as a platform, is important to assess for the development of new pharmacological approaches. However, whether the phenotypes of differentiated cells induced by direct reprogramming are affected by the source cell lines has not yet been examined. Therefore, the present study examined whether differences in fibroblast cell lines can affect the phenotypes of photosensitive cells induced by direct reprogramming.
Our data supported the possibility that the selection of cell lines might affect the phenotypes of differentiated cells, and that new pharmacological approaches for intractable retinal diseases could be designed at the level of each individual patient.

Cell Culture
Two strains of cultured human dermal fibroblasts were

Physiological Analysis
To assess photoresponses, we calculated the holding current and baseline noise before light stimulation for >6 s. When the holding current was larger than −500 pA or the standard deviation of baseline noise was larger than 7.0 pA, the data were excluded from the analysis. When the stepwise baseline shift started after light stimulation with a modest delay, we judged it as an artificial baseline shift and excluded it from the analysis. The photo stimulation-induced current around the maximum current amplitude was sampled for 1 s during the light stimulation. The peak amplitude was calculated as the mean of the sampled photo stimulation-induced current.
A peak amplitude larger than twice that of the baseline noise (standard deviation of baseline) before photo stimulation was classified as a "photo response". Total charge transfer across the membrane during light stimulation was integrated over time and normalized to the charge transfer per second 10 .

Statistical Analysis
We used the statistical software "EZR" 11 . Morphological data were analyzed by the Kruskal-Wallis test. After this test, statistics for individual data were performed using the Steel-Dwass test. Electrophysiological data were analyzed by Fisher's exact test.

Morphology of Induced Photosensitive Cells from Fibroblasts
To investigate the morphological changes in cells, first, we compared the basic morphological properties between fibroblast cell lines, Fib#1 and Fib#2. In non-infected fi-broblast cell lines, most cells were fusiform ( Fig. 1A and   C . 1E). In contrast, no difference was found in the number of primary processes between the cell lines (mean±SEM, Fib#1: 3.8±0.1, n=182, Fib#2: 3.5±0.1, n=158, P>0.05, Steel-Dwass test).
In CRNO-infected fibroblast cell lines, we observed very large cells with multiple primary processes (neurites) among the CRNO-infected cells (Fig. 1B and D). We also investigated whether the morphological properties of CRNO-infected fibroblasts differ between the two fibroblast cell lines. Contrary to the non-infected condition, the soma size of Fib#1 was significantly smaller than that of Fib#2, while no difference was found in the number of primary processes between infected cell lines.

Response Properties to Light Stimuli in Induced Photosensitive Cells
Next, we investigated whether physiological properties differ between cell lines of fibroblasts. In the present study, we considered cells with large sized soma as representative CRNO-infected cells and recorded the current from those large sized cells (Fig. 1B and D) as they responded to light stimuli 4 .

Light responses in retinal neurons are classified into
photoreceptor-type responses and intrinsically photosensitive ganglion cell (ipRGC)-type responses 12 . Although both types of light responses are mediated by cation channels (the main permeable ion is Na + ), their polarity is opposite. Light stimulation closes cation channels and hyperpolarizes photoreceptor-type cells, but opens cation channels and depolarizes ipRGC-type cells. In the present study, we set the holding potential close to the mem- to separate these two types of photoresponses. In our experimental condition, the closure of Na channels (photoreceptor-type response) is detected as an outward current, while the opening of Na channels (ipRGC-type response) is detected as an inward current (reversal potential of Na is +60 mV).

A detectable current was observed in both Fib#1 and
Fib#2 fibroblasts when photo-stimulation was found ( Fig.   2A and C). We observed both an outward current and an inward current. The outward current started to flow immediately and continued to flow even after the cessation of light stimuli (Fig. 2A). The inward current also started to flow without detectable delay and increased in amplitude quickly, and then increased in amplitude gradually until the end of light stimulation (Fig. 2C). The current flow decreased gradually after the cessation of light stim-uli and eventually returned to the baseline level. Light stimulation did not produce any detectable fluctuation of the baseline in non-infected cells in either cell line ( Fig.   2B and D). Since the distinct response to light was observed only once in most cells examined, we did not use the same cover slip for other trials.
To assess how many CRNO-infected cells responded to light, we calculated the peak amplitude and the total charge transferred across the membrane during light stimulation. Distinct "photo responses" were observed in 6 cells in CRNO-infected Fib#1 fibroblasts (n=20) and 17 cells in CRNO-infected Fib#2 fibroblasts (n=33) (Fig. 2E, open circle). Detectable "photo responses" were observed in no cells in the non-infected Fib#1 fibroblasts (n=15) and 1 cell in the non-infected Fib#2 fibroblasts (n=10) (Fig. 2E). The ratio of cells with "photo responses" was significantly greater in CRNO-infected cells than in non- while both an inward current and an outward current were observed in Fib#2 fibroblasts (Fig. 2F, open circle).
The distribution of total charge-transfer of cells with "photo responses" was similar to the distribution of current amplitude (Fig. 2G, open circle), confirming the plausibility of our criteria.
We also assessed whether the "photo responses" lasted after the light stimulation. We measured the amplitude of

Discussion
We showed that the type of fibroblast cell line might affect the physiological properties of induced photosensitive cells infected with CRNO genes by direct reprogramming. We found that "photo responses" in Fib#1 fibro-blasts were an outward current only, while "photo responses" in Fib#2 fibroblasts were an inward or an outward current. We also found a difference in the morphological properties of the photosensitive cells between the two fibroblast cell lines. This is the first report to describe that induced photosensitive cells produced by direct reprogramming can express a different phenotype.