Our results demonstrate that ImM10 conditionally immortalized Müller cells have some characteristics of retinal stem cells, in that they express many genes associated with neuronal stem cells and retinal progenitors and, after FGF2 and EGF growth factor stimulation, generate spheres that are morphologically similar to the neurospheres generated in culture by neural stem cells,40,41
retinal progenitors, and Müller glia.13,14,42
Ultrastructural analysis of ImM10-derived spheres revealed the presence of numerous macula adherens and gap junctions. Gap junctions are critical in neurosphere formation and in the dedifferentiation of embryonic stem cells43
and cortical neural progenitors44
and could play a role in the generation of Müller glia-derived spheres. When cultured in sphere-forming conditions, ImM10 cells expressed many genes typically associated with neural stem cells and retinal progenitors, including Nestin
, and Ccnd1
Interestingly, these genes are also expressed by ImM10 cells cultured in immortalizing growth conditions (J). Further, with the exception of nestin, we found no statistically significantly differences in mRNA expression of many genes typically coexpressed in retinal progenitors between growth and sphere cultures using quantitative RT-PCR. Gene expression in the ImM10 cell line is consistent with expression patterns in primary mouse Müller glia determined using serial analysis of gene expression,46
single-cell microarray analysis,50
Our results differ somewhat from reports of gene expression in other Müller glial cell lines. Spontaneously immortalized human Müller glia express Pax6
, and Notch1
mRNA under growth conditions (DMEM + 10% fetal calf serum), although only a fraction of cells are PAX6 immunopositive.14
Rat Müller cells cultured in DMEM/F12 with N2 supplement, EGF, and FGF2 generate spheres that robustly express SOX2, Nestin, and Musashi-1; however, expression was not reported for the same cells cultured in adherent growth conditions.15
Therefore, it is unclear whether sphere conditions specifically upregulate retinal stem cell genes in rat Müller glia or simply maintain expression of genes already transcribed in these cells.
Using identical media and growth factor stimulation on parallel cultures, we found that induction of neuronal gene expression is differentially regulated by growth on 2D versus 3D substrates. Mdk
, a heparin-binding growth factor that is important in neurogenesis, was expressed in all conditions but was upregulated only in 2D cultures. In zebrafish, mdk-a
are expressed in retinal progenitors and upregulated in proliferating Müller glia during the early stages of retinal regeneration.53 Sox3
also regulates neurogenesis and neuronal differentiation in zebrafish54
and was expressed only in 2D conditions. In contrast, Mef2c
was robustly expressed only in 3D conditions. Mef2c
is involved in early neuronal differentiation, and mouse embryonic stem cells that express a constitutively active form of Mef2c
differentiate into a nearly pure population of neurons.55
Consistent with the morphologic changes we observed in differentiation cultures, some genes associated with axon outgrowth, guidance, and synapse formation were also significantly upregulated in both 2D and 3D cultures (compared with growth conditions) consistent with a progression from neurogenesis toward differentiation. Among these, Bmp2
are involved in dendrite formation and neurite outgrowth56–58
and were upregulated only in 3D conditions. Nptx1
, a gene involved in synaptic refinement,35
was upregulated in 2D conditions, but was more robustly upregulated in 3D conditions. APOE is synthesized by cultured Müller cells and is secreted into the vitreous by Müller cells in vivo59
and was expressed in all conditions, though it was upregulated only in 2D differentiation cultures. Apart from its well-described role in cholesterol transport, APOE modulates long-term potentiation in the hippocampus through its involvement in NMDA and AMPA receptor regulation.60 Grin1
, the ionotropic glutamate (NMDA) receptor 1, was also upregulated in 2D conditions. In the rodent retina, NMDA receptor subunits are expressed primarily on inner retinal amacrine and ganglion cells.61
In addition to changes in gene expression associated with neural stem cells and neurogenesis genes in general, several genes specific to retinal neurons were expressed in differentiation conditions. The cone-rod homeobox transcription factor (Crx
) is critical to the development of photoreceptors62
and was detected in only cells in 3D cultures using both RT-PCR and immunohistochemistry. Unexpectedly, CRX immunoreactivity in 3D cultures was both nuclear and cytoplasmic. Because antibody penetration and subsequent removal of unbound antibodies from the 3D matrix was more difficult than in 2D cultures, one possible explanation for the cytoplasmic staining is increased nonspecific staining. We cannot exclude the possibility that at least some of the immunoreactivity reflects mislocalization of the CRX protein. Other studies of in vitro differentiation of retinal stem cells and Müller glia have shown aberrant cytoplasmic localization of proteins including Chx1014
regulatory targets were expressed in 2D cultures in the absence of detectible Crx
expression. Recoverin is a calcium binding protein expressed in photoreceptors that is involved in the inhibition of rhodopsin kinase and the adaptation to light.63
Recoverin was expressed at low levels by ImM10 in all conditions but was robustly upregulated in 2D differentiation conditions. Cells in both 2D and 3D conditions expressed the transcripts for m-cone opsin, but it was more strongly upregulated in 3D conditions. Upregulation of recoverin in 2D cultures in the absence of Crx
expression most likely reflects positive regulation by other transcriptional regulatory genes because, in the retinas of Crx
null mice, recoverin expression is downregulated but still detectable.64
Although no m-opsin expression is detected in Crx
null retinas by serial analysis of gene expression,65
the nuclear receptor TRbeta2 is a regulator of M-opsin.66
Therefore, the expression of TRbeta2 or other positive regulators of m-opsin could contribute to low levels of transcription.
Our culture conditions were modeled on a previously published study in which FGF2 priming followed by B27 differentiation resulted in the upregulation of photoreceptor markers in approximately 30% of retinal progenitors cultured from the neonatal (P0-P2) mouse retina.31
We observed the upregulation of some genes characteristic of rod photoreceptors in both 2D and 3D cultures but did not observe cells with the distinct morphology of photoreceptors. Further, although markers for multiple classes of retinal neurons were expressed, there was no obvious bias toward gene expression patterns associated with any single retinal cell type in either 2D or 3D conditions. Variability in the neuronal genes that are upregulated in Müller glia in vivo after retinal injury has also been reported. Injection of neurotoxic agents or the gliotoxic α-aminoadipate results in the upregulation of genes characteristic of late-born cell types (photoreceptor and bipolar cells) in a subset of Müller glia.8,11,67
In contrast, after intraocular NMDA injection, some Müller cells acquired amacrine cell phenotypes suggesting that Müller cells can also generate early-born retinal cell types.9
Consistent with this, ImM10 cells in 2D cultures upregulated melanopsin, which is expressed in a subset of retinal ganglion cells. The more robust generation of rod photoreceptors in vitro from neonatal retinal progenitors may reflect an intrinsic bias in progenitors isolated from a developmental stage when they are actively generating rod photoreceptors in vivo.
Mammalian Müller glia can upregulate genes characteristic of retinal neurons after injury in vivo or growth factor stimulation in vitro. However, in all studies published to date, only a subset of Müller glia adopt a neuronal-like morphology. ImM10 cells also showed a persistence of glial morphologies and a relatively low frequency of differentiation. We also found that some of the cells that upregulated neuronal genes, such as CaBP1
, retained a distinctly glial morphology. This is consistent with reports showing that after in vitro differentiation of human Müller glia, protein kinase C immunoreactivity was present in cells with a distinctly glial morphology.14
It remains to be determined whether the changes in gene expression observed in Müller glia in vitro represent aberrant expression of neuronal genes in glial cells or incomplete differentiation into neurons.
To our knowledge, there are no previous studies examining the effect of 3D substrates on Müller glia. We found that the general behavior of ImM10 Müller glia in RADA-16 hydrogels was similar to those cultured on tissue culture plastic. When embedded as single cells and treated with EGF/FGF2 sphere-forming medium, ImM10 cells generated spheres within the gels. When embedded as spheres and cultured in priming and differentiation conditions, cells would often completely migrate from the spheres and extend processes, similar to their behavior in 2D. Other researchers have shown that retinal progenitor cells isolated from the perinatal mouse retina can migrate into the network of pores within biodegradable scaffolds made of poly lactic-co-glycolic acid (PLGA) or poly-L-lactic acid (PLLA).19,20
In contrast, in preliminary studies using a thiol-modified sodium hyaluronate and gelatin-based hydrogel (Extracel; Glycosan Biosystems, Salt Lake City, UT), we observed minimal proliferation, migration, or process extension (data not shown). Similarly, when seeded onto glycerol sebacate polymers with precast 50-μm pores, most perinatal retinal progenitors remained within the pores and did not migrate into the dense surrounding polymer.22
Retinal progenitors cultured on micropatterned polycaprolactone films failed to form clusters and retained a circular or glial morphology, despite increased expression of recoverin and rhodopsin.68
Perinatal retinal progenitors cultured on PLGA/PLLA gels and then subsequently transplanted subretinally into rhodopsin null mice showed increased survival and expression of recoverin compared with cells transplanted without the gels. Differential migration likely reflects the physical and chemical structures of each type of polymer as well as differences in culture conditions and the types of cells.
It is notable that the RADA-16 hydrogels used in our studies were not modified to add specific adhesion molecules, attachment motifs, or growth factors, and both 2D and 3D cultures used identical culture conditions. Thus, the changes in gene expression can be attributed to the presence of the 3D matrix. Because largely unique sets of neuronal genes were upregulated in 2D and 3D cultures, we cannot conclude that one method is superior to the other for promoting neuronal differentiation of Müller cells. Future studies comparing the effects of different matrices in combination with specific modifications will be necessary to identify the optimal substrate to enhance the differentiation and promote the generation of specific retinal cell types. The ability to cast cells into the RADA-16 hydrogel could offer advantages for generating sheets of cells for transplantation, and our finding that cells can readily migrate within the gels would permit transplanted cells to integrate within the host tissue. For tissue engineering purposes, it may ultimately prove useful to develop combination scaffolds that incorporate patterned structures and chemical modifications to direct cell growth and provide specific growth factors or cell adhesion molecules.
Our results add to the mounting evidence that Müller glia express many genes characteristic of retinal progenitors and support the proposal that adult Müller glia could function as retinal progenitors or stem cells. However, the low numbers of differentiated cells that have thus far been generated from mammalian Müller glia, either in vitro or in vivo, remain insufficient for therapeutic applications. Development of cell replacement therapies will require additional research to identify and overcome the restrictions that prevent mammalian Müller glia from achieving the robust regenerative ability they have in lower vertebrates.