Stem cell engineering has opened a new avenue for repairing damaged nervous tissues. Following transplantation of cultured stem cells into eyes, they further integrate into the retinal microenvironment, and then proliferate and differentiate into target cells, to regenerate damaged neurons . This offers recovery and reconstruction of the retinal function, with opportunities to treat irreversible blindness ophthalmopathy.
Previous work by Amirpour N et al. has demonstrated that the injection of ESCs into the subretinal space of rats effectively alleviated photoreceptor cell degeneration and death . However, it is difficult to obtain ESCs because in vitro culturing is limited , posing a bottleneck for transplantation. Neural stem cells (NSCs) have been found in the embryonic nervous system and in certain parts of the adult brain. Due to continuous self-renewal and proliferative ability, these cells can be differentiated into specific neurons and glial cells. Recently, it has been reported that these cells were successfully integrated into the various layers of the retina [26–28]. The major challenge is how the NSCs differentiate into mature retinal cells. Some studies have shown that differentiation is associated with the growth environment of the cell [29–31]. In the present study, we isolated, cultured, and propagated mouse RSCs from E17 embryos. After extended to the 4th generation, the cells presented with phenotype of RSC. To further verify if these cells are stem cell and have characteristics of NSCs, we stained the stem cell marker Pax6 and NSC-specific marker Nestin [21, 22]. Cultured RSCs not only presented stem cell morphologies under phase contrast microscope, but also highly expressed Pax6 and Nestin, indicating that the cultured RSCs belong to the NSC family. Therefore, embryonic RSCs might be becoming prospective cells for retinal transplantation.
Considering the RSC as the most suitable seed cell, we further examined the feasibility of RSC transplantation for treatment of damaged retina. In general, there are currently two types of transplantation methods: subretinal space injection and vitreous cavity injection [6, 32]. Maintaining intraocular structure and preventing immune rejection is crucial for the survival of transplanted cells. In the case of subretinal space injection, it is often difficult to avoid blood retinal barrier disruption within the eyeball, which can cause severe swelling as well as degeneration and/or death of transplanted cells . The intravitreal injection is a simplest feasible way in clinical practice for intraocular medication, which allows the eyeball to remain intact and reduces the occurrence of damage to the retina barrier. Moreover, this method offers a clear field of view during operation [6, 32, 33]. Although both of these grafting methods are capable of integrating seed cells into the retina, some studies recently compared the two methods, showing that trauma induced by the subretinal injection is considerably greater leading to retinal detachment . Therefore, the intravitreal injection method is commonly preferred for delivery of medication or stem cell. In our study, we delivered BrdU-labeled RSCs into vitreous cavity for cell transplantation. Upon examination of the retina post-operation till 2 weeks no infections or bleeding were observed. Close observation of sectioned specimens revealed a complete intraocular structure with apparent anatomic arrangement. These findings suggest that intravitreal injection is a safe delivery way with less trauma for transplantation.
In order to gain an insight into the cell arrangement, we investigated the effect of transplanted RSCs on the retinal neuron composition. Retinal neurons are typically divided into three layers: ganglion cells, bipolar cells, and photoreceptor cells, from the inner to outermost layer, respectively . Photoreceptor cells convert light stimuli into nerve impulses, the bipolar cells transfer nerve impulses to ganglion cells, and the nerve impulses transfer through the ganglion cells nerve fiber to the optical center, which produces the visual . As the retinal ganglion cells are associated to nerve fibers, any mechanical damage to the optic nerve can block axoplasmic transport of the ganglion cells, leading to direct impairment of ganglion cell nutrition . The pathology of such retinal damage and visual function lesions are mimicked by ring clamping of the optic nerve .
Here, we firstly performed retinal damaged mice model by ring clamping of the optic nerve. 1 week after operation, H&E histological analysis showed diffuse edema in the retinal tissues, along with disordered and loose-arranged morphological changes of all layers. We then attempted to transplant the BrdU-labeled RSCs in close proximity to the ganglion cell layer, which allowed us to assess the effect of damaged optic nerves directly. Notably, the percentage of the BrdU-positive RSCs was greater than 90%, indicating that almost all the RSCs were labeled. Characterization of transplanted tissues was thereafter carried out after 2 weeks. Our results showed that the BrdU-positive cells were present in vitreous cavity, the retinal surface and the ganglion cell layer, indicating a successful transplantation of the RSCs and their entry into the damaged retina. This is further demonstrated by the presence of Pax6 and Nestin-positive cells in the retina, predominantly between the retinal ganglion layer and inner nuclear layer. Morphological evaluation also suggests that the transplanted RSCs were integrated into the host retina, implying its potential to substitute for the damaged cells. This observation is consistent with previous reports [36, 37]. However, differentiation of the transplanted RSCs was not distinguishable through light microscopy.
Several studies have shown that transplanted neural stem cells in retinas can partially differentiate into neurons [38–40], but generally depend on microenvironment of the host [41, 42]. In addition, it has been reported that this differentiation is also related to the host age. Li N et al. showed that the integration ability of stem cells transplanted into the vitreous cavity of rats decreased with the increase of host age . Nevertheless, this phenomenon may be associated with a range of other factors such as the existence of a large number of undifferentiated cells in the neonatal host, an imperfect barrier function, and some growth factors promoting cell migration . Furthermore, it has been shown that when the graft contains more mature cells it easily forms aggregates of cells or rosettes, which disrupt the integration of graft and host and thus prevent the transplanted cells from reconstructing the retinal function . However, embryonic cells or stem cells are not easy to form a rosette even with a high number . As such, it is evident that the purity of transplanted cells is extremely important to allow differentiation into functional target cells even if ESCs or embryonic RSCs are used.