In this study, we sought to overcome the growth limitations of human prenatal retinal neurosphere cultures by modifying a method originally developed to expand human prenatal cortical neurospheres [35
]. Previously described techniques for culturing human prenatal retinal cells maintained them as dissociated monolayers for at least seven [88
] or eight [38
] passages, but growth declined at later passages depending on culture conditions. Using our standard medium, which included EGF and FGF2, both dissociated and neurosphere cultures of human prenatal retinal cells failed to demonstrate significant expansion.
Given the importance of RPE during early neuroretinal development [5
] and its pro-proliferative effects on rodent and chick RPC cultures [5
], we hypothesized that RPE CM would improve prenatal human retinal neurosphere growth as well. However, the degree to which RPE CM could selectively halt retinal neurosphere volume loss and restore a robust growth pattern was surprising. This effect may be mediated by multiple soluble factors, some of which are likely not exclusive to RPE, as shown by the lesser growth-promoting abilities of cortical neurosphere and lens CM. It is also important to note that RPE CM could not improve growth of neurospheres derived from other human embryonic CNS tissues, and in fact had a detrimental effect on cortical neurosphere growth. These regional differences in response to RPE CM are being further investigated.
A recent examination of the growth factors and other proteinaceous components secreted by RPE cultures revealed a number of molecules with pro-proliferative activity in the developing brain and retina, including vascular endothelial growth factor and cystatin C [33
]. In addition, RPE CM contains high levels of pigment epithelium-derived factor [33
], a molecule that promotes cell survival in the retina [89
] and neural stem cell self-renewal in the subventricular zone of the brain [91
]. Inorganic compounds present in RPE CM may also play a role in the expansion of retinal neurosphere cultures, since CM protein fractions restricted to elements less than 3-kDa independently sustained modest neurosphere growth. One such candidate compound is ATP, which has recently been shown to support mouse RPC growth [20
]. Efforts are ongoing to identify the RPE CM factors involved in the observed retinal neurosphere growth enhancement.
While the secreted RPE factor(s) responsible for retinal neurosphere growth remain to be delineated, present evidence does suggest a mechanism for the effect. Our results show that RPE CM confers mitogen responsiveness to retinal neurospheres, a property that neurospheres from other CNS regions innately possess [41
]. The rapid phosphorylation of CREB following CM challenge suggests that RPE CM likely acts in part by potentiating existing cellular EGF and FGF2 signaling pathways and/or relieving potential inhibitory influence(s) present in self-conditioned retinal neurosphere medium.
Although RPE CM is effective in promoting the long term expansion of retinal neurosphere cultures, it does not prevent the progressive cell fate restriction observed in its absence [35
]. Specifically, in both RPE CM-treated and untreated cultures, retinal neurospheres invariably lost their neurogenic potential after 2–3 months in vitro
and became increasingly gliogenic. This pattern of cell fate determination is reminiscent of normal mammalian cortical and retinal development, during which progenitor cells initially give rise to long projection neurons, followed by interneurons and/or photoreceptors, and finally glia [6
]. In the developing retina, Müller cells are the sole type of glia derived from RPCs [6
] and have been shown to retain progenitor characteristics after differentiation [65
]. However, the absence of mature markers suggests that our long term cultures do not consist of fully differentiated Müller glia. Other GFAP-positive glia found in the retina originate from the optic nerve, but these astrocytes are unlikely to be present in our cultures given the gestational ages used [38
], the method of dissection [35
], and the gene and protein expression profile of the cells [10
An examination of the transcription factor profile of undifferentiated, long term cultures of retinal neurospheres revealed a potential cause for their loss of neurogenic potential. Pro-neural, bHLH transcription factors such as Mash1 and NeuroD were absent by PCR, whereas transcription factors favoring Müller glia production (Hes1 and Rx) were present [6
], along with a host of genes and proteins expressed by neural and retinal progenitors. The expression of Hes1 in all of our cultures was particularly intriguing, since it also plays a prominent role in the maintenance of a proliferating pool of retinal progenitors [6
Taken together, our findings suggest that the fate potential of long term retinal neurosphere cultures is governed in large part by intrinsic limitations rather than extrinsic influences. Consistent with this hypothesis, culture modifications that enhanced neurogenesis in rodent RPCs failed to alter cell fate potential in late passage human retinal neurospheres [66
]. Therefore, we misexpressed the pro-neural bHLH transcription factor Mash1 in an attempt to overcome Hes signaling and push the hRPCs toward a neuronal fate. Mash1 was chosen because of its demonstrated efficacy in rat [71
] and human (unpublished observations) embryonic cortical neurosphere cultures and its role in neuron and photoreceptor production in the retina [11
]. Although lenti-Mash1 infected hRPCs were immunonegative for photoreceptor-specific proteins, they did express a number of markers indicative of inner retinal neurons, including ganglion cells, amacrine cells and bipolar cells. However, the majority of lenti-Mash1 infected cells remained nestin-positive and failed to demonstrate characteristics of either neurons or glia. A possible explanation is that low levels of Mash1 are sufficient to halt proliferation and thwart default gliogenesis in our hRPC population, but higher levels are needed to restore neurogenesis [71
]. Further manipulation of the transgene expression levels may improve the yield of neurons from these cultures.