We have previously used a defined SFEB/DLFA method of ES cell differentiation to successfully differentiate all retinal cell types (9
). Our present study used this protocol to determine whether mouse ES cell-derived photoreceptor precursors have the ability to migrate and integrate into the adult retina as efficiently as freshly dissociated retinal cells derived from an equivalent developmental stage in vivo
. We showed the efficient generation of early retinal progenitors and the correct birth order of retinal cell types in accordance with in vivo
retinal neurogenesis, using an optimized version of this ES cell differentiation protocol. We generated populations in which all the cell aggregates contained Crx positive cells (≤ 65 % of cells Crx+
) by day 24 as shown by immunocytochemistry and increased numbers of Crx transcripts by day 28, as demonstrated by qPCR, with similar numbers of transcripts compared with the early postnatal retina (P0). However, following the transplantation of this retinal cell population no integrated ES cell-derived photoreceptors were observed.
Following the scale up of the ES cell differentiation protocol to isolate populations for transplantation we found very few Nrl expressing cells. This may be due to several reasons including increased concentrations of inhibitory factors derived from other retinal cell types, such as ciliary neurotrophic factor (CNTF) produced by astrocytes and glial cells (26
), and previously shown to inhibit rod differentiation (30
) and to block Nrl
). In vivo
retinal development involves gradients of secreted signaling molecules, such as FGF2 and Shh (33
) that may not be present in the adherent ES cell-derived cultures. The lack of correct cell-cell contact, neural epithelial organization and polarity that are present in the in vivo
retina are not simulated in the 2D adherent culture environment, and may account for the reduced differentiation observed (34
). It was not possible to determine whether further time in culture increases the number of Nrl+
precursors as the survival of the cultured cells was compromised from day 32 onwards, with morphological deterioration by day 34. However, the majority of rod photoreceptors were born at day 20 of culture and mature photoreceptor markers were starting to be expressed at day 28 with maintained expression until day 32 and there was little indication of increased Nrl expression at this later stage.
The presence of tumors following ES cell and ES cell-derived transplantation to the eye has been shown previously in several other studies (36
). In transplantations to the brain, selection of Sox1.GFP+
cells and culture prior to transplantation attenuated tumor formation (40
). Here we found that despite selecting Rax.GFP+
retinal progenitors prior to transplantation there was still a risk of tumor formation from ES cell-derived cultures. However, following selection and extended retinal differentiation time, the percentage of transplants that presented tumors was greatly reduced. Further differentiation partially negated the need for FAC-sorting as any remaining progenitor/stem cells differentiated in vitro
prior to transplantation. This is important if mixed populations are to be transplanted. However, the most efficient method for the transplantation of freshly dissociated photoreceptors is by FAC-sorting the relevant Nrl+
cell population prior to transplantation. This is also likely to be the case for ES cell-derived retinal cells and the future selection of photoreceptor precursors by reporter expression or surface marker combinations may enable the improved quantification and isolation of this population within the culture (41
When assessing whether transplantation of ES cell-derived retinal cells resulted in the integration of new photoreceptors, we observed a small number of false positive results that we conclude were due to AAV2/9 CMV.GFP transduction of host photoreceptors. Like the host cells in the recipient knockout models, these cells did not express photoreceptor markers. It is important to note that even following several changes of media in the days preceding transplantation, extensive washing prior to dissociation and FAC-sorting of the cell population it still may be possible that some residual infective virus particles may be shuttled with the cells. Using ES and iPS cell lines that contain endogenous reporters would avoid the problem of false positives due to viral vector. Increased cell death in the transplanted cell population, increased gliosis of the host retinas or increased innate immune responses in the recipients were all excluded as possible explanations for the lack of integration of the ES cell-derived retinal cell populations. We propose that the most likely explanation for the lack of integrated cells was the presence of low numbers of optimally staged ES cell-derived precursors in the cultures. We have previously shown that Crx.GFP expressing embryonic stage cells from the developing retina show at least a ten-fold lower integration efficiency than those from the postnatal retina (42
), so the Crx+
ES cell-derived populations may show similarly low transplantation competence.
Our results differ from those of Lamba et al
. who have reported the integration of human ES cell-derived photoreceptors into the neo-natal and adult mouse retina (13
). This disparity may be due to inherent differences between the efficiency of retinal differentiation of human and mouse ES cells in vitro
. In the study by Lamba et al
., 15 % of human ES cell-derived cells were reported to be Nrl+
following 3 weeks of retinal differentiation culture, and therefore the transplanted cell population may have contained greater numbers of integration-competent photoreceptor precursors (13
). However, one similarity with our study is the use of viral vectors to label the transplanted cell population, which we demonstrated could lead to false-positive results. In these circumstances, as shown in our study, it is essential to use additional, unambiguous markers to identify transplanted cells.
Our study indicates that further investigations are required to demonstrate convincingly that ES cell-derived cell cultures can give rise to new mature functional photoreceptors. We conclude that the defined SFEB/DLFA method of ES cell differentiation which relies on a 2D culture method for retinal cell differentiation could not be scaled up sufficiently to produce large numbers of transplantation competent photoreceptor precursors. These data suggest that although this culture method generated Crx-expressing photoreceptor precursors, the majority of Crx+
cells were impaired/delayed in their differentiation pathway and failed to activate the Nrl driven rod differentiation pathway. After transplantation into the adult retinal environment we found no evidence that the ES cell-derived cells were able to further differentiate and exhibit outer segments. New methods such as the 3D differentiation method recently published by Eiraku et al.
may enable transplantation competent photoreceptor precursor cells to be generated in vitro
to better facilitate transplantation studies (18
). This recent work has shown the generation of optic cup-like structures that give rise to layered retinal structures containing high numbers of photoreceptors. This new method of in vitro
differentiation may greatly improve the transplantation potential of ES cell-derived cells as they differentiate in a tissue niche, which may help to promote full differentiation to the photoreceptor precursor stage equivalent to early postnatal development.