A large body of literature evidence has confirmed that adult mammalian and primate retina are amenable for cell replacement therapy. Transplantations of human fetal retinal progenitors
34,35 or photoreceptor precursors
12 are shown to preserve or improve visual functions in animal models of retinal dystrophy. However, a reliable and renewable source of donor cells such as the hESCs and iPSCs are being intensely explored for adopting them in cell replacement therapies. As mentioned earlier, many reports have shown that retinal cells derived from hESCs are amenable for scaling up and are effective in delaying disease progression and in improving visual functions in preclinical animal studies.
The results of the present study confirm the retinal differentiation potential of the hESC line, BJNhem20. Using the protocol described here, it is possible to efficiently differentiate and enrich RPE cells and NR progenitors and expand them enough for further downstream applications. Although complex cocktails of culture components have been reported to drive retinal differentiation, our protocol involves an initial EB formation and short-term culture in the presence of Noggin and N2 supplement to drive neuro-ectodermal differentiation. Subsequent differentiation in B27-supplemented conditions promoted eye-field specification within 1 month. Manual isolation of these mildly pigmented eye-field–specified clusters ensures early enrichment of retinal progenitors. These early-stage retinal progenitors expressed Nestin, GFAP, Pax6, Chx10, Rx, and Mitf. Adherent culture of isolated retinal progenitors gave rise to both RPE and NR patches. Expansion of isolated RPE cell patches, their maturation, pigmentation, and maintenance of proper cell morphology was enabled by culturing them in the presence of ActivinA and ROCK inhibitor. The mature RPE cells were highly pigmented, polarized cells with apical microvilli and hexagonal morphology, and expressed RPE65, ZO-1, VEGF, and PEDF proteins at appropriate levels. Similarly, bFGF and DKK1 treatment enabled the expansion and maturation of NR cells that expressed Crx, recoverin, rhodopsin, cone opsin, and other NR markers.
Recent reports on the clinical outcomes of FDA-approved, phase I/II trials have established the safety and tolerability of subretinal transplantations of hESC-derived RPE cells in the treatment of patients with Stargardt's macular dystrophy and dry AMD.
17,18,36 It is well known that the eye is an immune-privileged site, being established by the barrier functions of RPE cells and the endothelial cells of retinal vasculatures. The early observations and outcomes of this trial confirm that in spite of being an allogeneic cell source, the hESC-derived RPE cells transplanted in the subretinal space of the patient's eye did not elicit any significant immunologic response. The reports also revealed that there were no signs of hyperproliferation, tumorigenicity, or ectopic tissue formation. Fundus photographs and optical coherence tomography images of transplantation sites of the retina in treated eyes have confirmed that the RPE grafts survived, proliferated/expanded in vivo for up to 37 months post transplantation, and contributed to marginal improvements in visual parameters. These developments were encouraging to consider BJNhem20-derived retinal cells for possible preclinical and clinical applications.
As a next step toward clinical translation, it is important to adapt early-passage cells into feeder-free and xeno-free culture conditions under a current good manufacturing practice (cGMP) work flow. Also, the retinal differentiation steps described in the study could be easily adapted to GMP requirements using clinical grade, xeno-free reagents. To ensure the safety of hESCs and their derivatives, it is important to reconfirm their pathogen-free status (bacteria, fungi, mycoplasma, and viruses, such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, and syphilis) using appropriate GMP-compliant testing methods. It is also important to ensure that the enriched RPE cells are free of undifferentiated cell contaminants by testing them for the absence of tumor formation when transplanted in immune-compromised mice models. Finally, a preclinical safety and efficacy study in retinal dystrophic rodent models would ensure the biological activity and functional relevance of ESC-derived retinal cells.
Although these are clear and obvious requirements for future clinical considerations, this report highlights the retinal differentiation potential of the hESC line, BJNhem20. The retinal derivatives and their progenies could serve as in vitro models in basic research and in pharmacologic drug screening and testing. A cGMP-compliant cell preparation could serve as valuable allogeneic donor cells in the treatment of patients with various forms of retinal dystrophies.