The ability to isolate tissue-specific progenitor populations from live, differentiating human pluripotent stem cells enhances their scientific and clinical utility. Beyond supplying a potentially expandable and relatively uniform cell source for transplantation, such populations facilitate in vitro studies of human cellular development and disease.
The development of the vertebrate retina can be traced morphologically, beginning with the evagination of OVs from the lateral aspects of the primitive forebrain. At this stage, chick OVs can be dissected from surrounding tissues and cultured short-term as explants, during which time they maintain their characteristic vesicular structure41
. However, a similar in vitro hallmark of early retinal populations had not been described in human pluripotent stem cell cultures prior to this report. OV-like structures isolated from hESCs and hiPSCs were anterior neuroectodermal in origin, expressed early retinal markers, and gave rise to mature retinal cell types, confirming their status as RPCs. In addition, the order of appearance of retinal cell types from OV-like structures approximated the conserved cell birth order found during vertebrate retinogenesis10,12
While OV-like structures resemble true OVs in some respects, the similarities between them are limited. Unlike true OVs, hESC- and hiPSC-derived OV-like structures did not invaginate to form bilayered cups using the minimal culture conditions we employed. Instead, they “filled in” and became relatively unorganized, presumably as a result of continued RPC proliferation in the absence of appropriate signaling cues. In addition, during normal vertebrate OV development, production of Chx10+ neuroretinal progenitors is restricted to the distal OV, whereas the proximal OV remains Mitf+ and yields RPE29,30
. This segregation of the neuroretinal and RPE domains is governed in part by reciprocal effects of FGFs and TGFβ superfamily members produced by surface ectoderm and extraocular mesenchyme, respectively41,52
. Similarly, canonical Wnt signaling has been shown to promote RPE generation while antagonizing formation of neuroretina53
. The lack of RPE production in isolated OV-like structures, which are cultured in defined medium without these factors, suggests an intrinsic bias toward a neuroretinal fate. In support of this hypothesis, we found that hESC-derived sphere cultures endogenously expressed FGFs along with inhibitors of TGFβ superfamily and Wnt signaling17
. In the present study, we further showed that this neuroretinal bias could be partially overcome via the addition of the TGFβ superfamily member Activin A. An analogous situation has been described in the chick model, where OV explants grown in isolation failed to generate RPE, but did so when exposed to Activin A41
Beyond adopting distinct morphologies and expression profiles, we also demonstrated that retinal cells derived from human pluripotent stem cells possessed important functional properties. Patch clamp recordings of RECOVERIN+ cells revealed a current-voltage relationship consistent with mammalian photoreceptors31–35
, as well as robust depolarization in response to administration of cGMP analogs. The latter finding is particularly intriguing, since cGMP is a critical second messenger of the phototransduction cascade38
, responsible for modulating the circulating ‘dark current’39
. While RGCs can also depolarize after cGMP stimulation54
, all cGMP-responsive cells tested in our cultures were RECOVERIN+ and lacked long neurite projections characteristic of RGCs. Therefore, the identity of the recorded cells was most consistent with photoreceptors. To document functionality of hESC- and hiPSC-RPE cells in a novel manner, we examined their response to ATP, an extracellular signaling molecule linked to the generation of IP3
and release of intracellular calcium55
. Through this mechanism, ATP is proposed to regulate ion and fluid flux across the RPE43
, among other critical functions. Exposure of hESC- and hiPSC-RPE to ATP resulted in rapid and reversible increases in intracellular calcium, analogous to responses elicited from prenatal human RPE cultures.
While the isolation of OV-like structures from differentiating hESCs provides a dynamic tool for the study of human retinal development, it may prove more useful for hiPSC research. Multiple reports have detailed variability in fate potential between hiPSC lines7,17,45,47
, some of which displayed little capacity to yield retinal cell types. In the present study, we found that OV-like structures were generated by every differentiating hiPSC line tested, although sometimes in very low abundance. Even so, their distinctive appearance allowed them to be isolated and cultured independently. It was also noted that retinal differentiation efficiency across hiPSC lines correlated with early endogenous expression of factors (DKK1 and NOGGIN) known to influence anterior neural fate specification. We have since used this information to quickly and inexpensively screen new hiPSC lines for competency to produce OV-like structures.
As methods of hiPSC production and differentiation continue to improve, so will the potential to apply this technology to model and treat human diseases. Inherited RDDs appear particularly amenable to hiPSC modeling, since many exhibit gene-specific, cell autonomous features that can be tested in vitro. In addition, broad sequelae common to multiple inherited RDDs, such as loss of rods, can be monitored in culture and used to assess drug efficacy5
. For the present study, we chose to derive hiPSCs from a patient with GA, an RPE-based RDD for which a simple assay exists to measure activity of the affected gene product, OAT27
. This enzyme is expressed in multiple tissues, but has particularly high activity in RPE27
, which may explain why clinical manifestations of GA are largely restricted to the retina. We confirmed that (A226V)OAT hiPSC-RPE had very low OAT activity, and further showed that these cells were exceptionally responsive to treatment with vitamin B6
, a pharmacological agent known to reduce serum ornithine levels and improve fibroblast OAT activity in a small cohort of GA patients. However, direct tests of vitamin B6
efficacy in RPE derived from a living GA patient have heretofore been unfeasible. We found that 600 μM vitamin B6
, but not 200 μM, completely restored OAT activity in (A226V)OAT hiPSC-RPE, while higher concentrations showed no additional benefit. These findings are potentially important since excessive vitamin B6
supplementation can cause painful and ultimately irreversible neurological side effects56
Perhaps the most imminent clinical role of hiPSC technology is in pharmacologic testing of rare, genetically heterogeneous diseases that 1) display variations in drug efficacy between individuals and 2) affect tissues that cannot be routinely or safely biopsied. Even when surrogate tests of drug response are available, patients may be better served by direct testing of cell types targeted by the disease. For example, information from the repository where we obtained the GA fibroblasts indicated that the donor was vitamin B6
unresponsive. However, other patients with the A226V OAT mutation51
, as well as the donor’s own hiPSC-RPE, were shown to respond to such treatment. It is therefore possible that the patient would have had a therapeutic response to vitamin B6
at the RPE level, but because efficacy was determined by less sensitive means, the treatment was erroneously deemed unbeneficial.
A more distant application of hiPSCs may be as a source of autologous donor cells for replacement therapies. To serve in this capacity, repair of disease-causing gene defects will likely be necessary. Recently, Howden et al. corrected the OAT
mutation in our patient using BAC-mediated homologous recombination25
and showed that this process did not introduce new mutations into the hiPSC genome. Here, we further demonstrated that OAT activity was fully restored in the gene-corrected hiPSC-RPE. To our knowledge, this is the first report of functional correction of disease-specific hiPSCs using both pharmacologic and genetic approaches.