The success of stem cell therapy is highly dependent on the ability of donor cells to migrate into the desired location, to survive after transplantation, and to differentiate into retinal cells to restore retinal function. Recent researches have shown that several cell populations may be considered as potential sources. These include fetal stem cells, pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) and adult stem cells.
Fetal stem cells
Fetal stem cells are fetal retinal cells, at the exact developmental time when these cells are born and about to form intrinsic connections. Previous studies have shown that, before the formation of synaptic connections, retinal ganglion cells can regenerate after axotomy and navigate through the optic chiasm [
8]. It has been proposed that immature photoreceptors might also have the capacity to reconnect themselves to the central neural system (CNS) after transplantation. Fetal retinal progenitor cells (RPCs) derived from a range of mammalian species, including rats [
9,
10], pigs [
11], and humans, [
12] have been tried. It has been shown that rodent fetal RPCs are able to propagate extensively, expressing photoreceptor markers. Transplantation of fetal RPCs resulting in the survival and differentiation of the grafted tissue has been proven to be associated with behavioral benefits in retinal dystrophic recipients [
13,
14]. Fetal neurons appear to show higher survival capacities than adult neurons [
15]. For human fetal-derived retinal cells, Young [
12] isolated proliferating human retinal progenitor cells (hRPCs) from 10th to 13rd week of gestation, and demonstrated that they could be expanded in tissue culture. However, their proliferating capacity was weak, and population declined quickly. Recently, Aftab et al. [
15] have shown that donor tissues taken from 16th to 18th week of gestation give the longest in-vitro survival time, and the highest number of cells. After transplantation, these cells were integrated into the recipient retina, and differentiated into rhodopsin positive cells. This result supported the potential of hRPC transplantation for degenerative diseases. Nevertheless, ethical issues still exist, and the supply of such cells is still limited.
Embryonic stem cells and induced pluripotent stem cells
An alternative is to use embryonic stem cells and induced pluripotent stem cells (ESC/iPSC). ESCs/iPSCs have a great potential to differentiate into any of the 200 or more adult cell types. Hence ESC/iPSC provides potentially unlimited cell sources for the generation of retinal cells. In-vitro differentiation of ESC/iPSC into functional retinal cell types is achievable by defined step-wise protocols [
16‐
19]. ESCs could be induced to differentiate into eye-like structures that contained cells with properties of crystalline lens, neural retina, and RPE [
20]. Further studies have indicated that cells from these eye-like structures could be further differentiated into RGCs when transplanted into the vitreous of an injured adult mouse retina [
21]. Recently, the success of defined differentiation of human ESC-derived RPE cells (hESC-RPE) has been reported [
22]. Following transplantation in animal models, restoration of vision had been reported and no tumor formation was seen [
19,
23]. In 2010, the FDA approved the first clinical trial using hESC-RPE for the treatment of dry AMD and Stardgart’s disease (STGD) in humans. Hopefully results will be available in the near future. The main advantage over adult-derived RPE cell lines is the ability to produce differentiated RPE cells in vitro, which is less immunogenic.
Transplantation of hESC-derived RPE cells has proved to be a milestone in clinical therapeutics. Nevertheless, its use is still limited by ethical controversies and the risk of rejection. Induced pluripotent stem cells (iPSC) offer an alternative cellular source for patient-specific treatment without the risk of rejection and ethical problems [
24,
25]. Nonetheless, clinical application of iPSCs is limited by the risks of proviral integrations and potential insertional mutagenesis during delivery of reprogrammed factors using virus. To overcome these issues, efforts toward the generation of “clinical grade” iPSC have been proposed. Recently, the reprogramming technologies in iPSC generation have been rapidly improved by the use of chemicals, plasmids, synthesized mRNAs, and direct protein delivery [
26‐
29]. In the future, transplantation of photoreceptors with or without RPE cells derived from these sources provides enormous potential for treating retinal degenerations. Personalized treatment strategy is potentially possible with the use of iPSCs, assuming that the risks associated are minimized.
Adult stem cells
It is known that lower vertebrates, such as teleosts or amphibians, have the ability to regenerate new retinal neurons throughout life, from a region called the ciliary marginal zone (CMZ) [
30,
31]. It was also thought that the adult mammalian ciliary body (CB) might harbor retinal stem cell. In 2000, two independent groups discovered that the ciliary epithelium (CE) of the murine eye contains multipotent retinal stem cells [
32,
33]. It was shown that single pigmented cells from the CE of mouse retina could clonally proliferate in vitro and form sphere colonies. These cells have the ability to be induced into retinal-specific cell types, including rod photoreceptors, bipolar neurons, and Müller glia.
Similar multipotent retinal stem cells were later identified in other mammalian species, including pigs and humans [
34,
35]. These cells were proliferative, but to a lesser extent than fetal or ESC-derived retinal stem cells. When these cells were transplanted into adult mice, new photoreceptors were induced [
35].
Another source of retinal stem cells was later discovered within the iris epithelium by Haruta and colleagues in 2001. The iris epithelium might harbor discrete heterogeneous populations of cells endowed with innate neural stem cell properties, including the ability to differentiate into retinal specific neurons [
36,
37].
After the discovery of adult retina-specific stem cells, a number of laboratories have sought to expand numbers of such adult retina-specific stem cells and optimize sub-retinal differentiation. However, there are several obstacles to the use of such cells. Firstly, the percentage of actively proliferating cells in the CE is very few (<2%) [
38]. Secondly, self-renewal and proliferation rates would decrease gradually with subsequent passages [
35,
38]. Thirdly, there may be a risk of tumour formation, as reported by Djojosubroto [
39] in a recent study. Furthermore, Gualdoni [
40] found that the expansion of CE-derived cells quickly led to the loss of retinal progenitor cell markers and hence reduced the potential of photoreceptor differentiation. Further investigations are needed to delineate the intrinsic mechanisms governing adult stem cell self-renewal and differentiation, as well as genetic stability.
Other adult stem cells have also been reported to be capable of inducing retinal regeneration. These include neural progenitor cells (NPC) [
41,
42], hematopoietic stem cells (hSC) [
43], and mescenchymal stem cells (MSC) [
44]. NPCs have been shown to promote the recovery from retinal injury and to express retinal phenotypic neurochemical markers [
45]. However, reports have shown that NPC lacks the ability to differentiate into mature retinal neurons [
46]. Furthermore, the shortage of adult NPC sources has further limited its application.
Autologous transplantation using hSC or MSCs has the advantage of reducing the risk of rejection and avoiding ethical controversies. Using retinal ischemia-reperfusion models, anatomical integration has been reported by intravitreal injection of hSCs [
43,
47] and MSCs [
48]. Animal studies have demonstrated that MSCs is capable of integrating into the ganglion cells and nerve fiber layers. Due to the fact that MSC derived from an elderly donor has limited functions, MSC derived from human embryonic stem cells or iPSCs serves as an alternative source [
49,
50].
Functional retinal differentiation from hSCs or MSCs is still highly debatable. More evidence has suggested that improvements with the use of adult hSCs or MSCs may actually be attributed to the secreted neurotrophic factors and anti-inflammatory cytokines in situ, instead of direct functional retinal differentiation [
44,
51].
Retina-specific cell types can be derived from various cell sources. Different cell sources and important growth factors and chemical modulators used to promote retinal cell differentiation are summarized in Table
1.
Table 1
Different cell sources and important growth factors/ chemical modulators used to promote retinal cell differentiation
Fetal stem cells |
Retinal progenitors (r) | EGF, FGF2, heparine | Photoreceptors | |
Neural retina progenitor cells (r) | FGF2 and NT3 (removal from medium) | Glial cells, neurons expressing rhodopsin, calbindin, calretinin | |
Progenitor cells neural retina (porcine) | CNTF and no EGF and bFGF | Photoreceptors | |
Human retinal progenitor cells | NT3, FGF2 | Retinal cell (cell culture) | |
Retinal progenitor cells (m) | EGF | Mature neurons, rhodopsin, or cone opsin | |
Photoreceptor precursors (m) | Transplantation of cells into immature retina | Rod photoreceptors, synaptic connections | |
Retinal progenitor cells (h) | Transplantation of cells into 16 to 18 weeks G.A. B6 mice | Photoreceptors | |
ESC and iPSC |
ESCs (h) | Stepwise treatment with defined factors | Photoreceptors and RPE | |
ESCs and iPSCs (h) | Casein kinase I inhibitor, ALK4 inhibitor, the pho- kinase inhibitor | Retinal progenitors, retinal pigment epithelium cells and photoreceptors | |
iPSCs (h) | No bFGF | RPE (cell culture) | |
ESCs(h) | KOM, nicotinamide ,TGF | RPE (cell culture) | |
ESCs (m) | bFGF, Dex, cholera toxin | A structure consisting of lens, neural retina, and pigmented retina(tissue culture)(cell culture) | |
ESCs(m) | NMDA-treated eyes | Eye-like structure | |
ESCs(h) | bFGF, xeno-free | RPE (tissue culture) | |
ESCs(m) | No LIF, retinoic acid | Neural progenitors , retinal cells | |
iPSCs(h) | KOS, zfbFGF, taurine, triiodo thyronin, hydrocortisone | RPE | |
Adult stem cells |
Dissociated cells from the RPE and the NR (m) | EGF, FGF2 | Rod photoreceptors, bipolar neurons, and Müller glia | |
Adult iris, pars plana, and ciliary body progenitor cells | FGF2 | Neurons and glia | |
Pars plicata and pars plana of the retinal ciliary margin progenitor cells(h) | FGF2, heparin, EGF | Photoreceptors | |
Multipotent cells within the IPE of postnatal and adult (r) | bFGF | Neural retinal cells, RPE, photoreceptors (cell culture) | |
Adult hippocampus-derived neural progenitor cells (r) | N2, bFGF | Retinal neurons | |
Hematopoietic progenitor cells (m) | SDF-1α | RPE | |
Hippocampus-derived neural stem cells (r) | N2, bFGF | Neurons and glia | |
Adult CD90 + MSC (r) | activin A, taurine, and EGF | Rhodopsin, opsin, recoverin | |
UCB-MSCs (h) | TGFβ, CNTF, NT-3, BDNF | RGCs (superior colliculus) | |
Ciliary body (m) | bFGF, GDNF | Photoreceptor, bipolar cell | |
Iris(r) | FGF2 | Rod photoreceptor | |
Adult bone marrow stem cells
Cells of bone marrow origin have also been used for retinal regeneration. Bone marrow contains subsets of non-haematopoietic lineages, which are capable of multi-lineage differentiation into cells of non-haematopoietic capabilities. These include mesenchymal, endothelial, and very small embryonic/epiblast-like stem cells (VSEL). These cells proliferate and act in response to tissue injury or damage. Although most of these cells are organ-restricted, some appear to retain multipotential capacities [
52]. In a mouse model, Li [
53] showed that adult bone marrow-derived stem cells (BMSC) could be induced into RPE lineage in vitro. When infused back in vivo, these BMSC-derived RPE cells can home onto the focal areas of RPE damage, and form a monolayer on Bruch’s membrane.