Abstract
purpose. To investigate the differentiation of rat neural stem cells (rNSCs) into cells of retinal pigment epithelial (RPE) lineage both in vitro and in vivo, after subretinal transplantation into normal rats and in a sodium iodate (NaIO3) model of RPE loss.
methods. rNSCs prepared from the cortex of embryonic day (E)14 Fisher F344 rats were cocultured with different concentrations of vasoactive intestinal peptide (VIP), adult rat RPE cells, or neurosensory retina (NSR) for 5 days. Cell morphology and expression of RPE-specific markers (cytokeratin, CD68, microphthalmia-inducing transcription factor [MITF]) were studied. Additional antibodies used to characterize the rNSCs were markers for stem cells (nestin), immature neurons (βIII-tubulin), astrocytes (glial fibrillary acidic protein [GFAP]), and oligodendrocytes (Rip). In in vivo studies, 106 green fluorescent protein [GFP]–labeled rNSCs were injected subretinally in either normal adult Lewis rats or NaIO3-treated rats (70 mg/mL NaIO3 administered intravenously 7 days before transplantation).
results. In vitro VIP-treated rNSCs changed from round cells to glia-like cells with processes that stained for both GFAP and nestin. In addition, small clusters of flattened, polygonal cells with an epithelial-cell–like shape that stained for cytokeratin and CD68 were observed. Coculture of rNSCs with RPE cells, but not with NSR, also led to cells of this phenotype. After transplantation, nestin+ and GFP+ rNSCs were visible subretinally as a transplant. In addition, more than 50% of transplanted rNSCs were cytokeratin+ and CD68+.
conclusions. Very few rNSCs differentiate in vitro into epithelial-like cells that express RPE-specific markers. In vivo, this differentiation is remarkably enhanced after subretinal engraftment. Thus, transplantation of NSCs into the subretinal space may be a therapy for retinal diseases involving an RPE abnormality.
Stem cells have the capacity to self-renew as well as to give rise to specialized cell types. They are uncommitted and remain uncommitted until they receive a signal to develop into distinct cell types.
1 Besides embryonic stem cells that are pluripotent and derived from the blastocyst,
2 adult stem or precursor cells, which have a more limited potency to give rise to specialized cells, are found in different somatic tissues including the central nervous system (CNS) (see review
3 ). Irrespective of their origins, stem and precursor cells are multipotent in differentiation and transdifferentiation, responding to stimuli both in vivo and in vitro.
4 5 6
Neural stem cells, expressing the neural intermediate filament nestin,
7 are found in regions of continued high-rate neurogenesis-like dentate gyrus or subventricular zones throughout life.
8 They can grow in vitro in the presence of defined growth factors (e.g., basic fibroblast growth factor [bFGF] and epithelial growth factor [EGF]
9 ) and can be directed to differentiate into neuronal or glial cell types.
10 11 Furthermore, the microenvironment, healthy or pathophysiological, may play a pivotal role in the fate of the neural precursor cells after transplantation.
12 13 Neural stem cells appear to have a wide differentiation potential, in that it is possible to turn these cells into a range of blood cell types in vivo.
5 However, the question remains of whether this transdifferentiation is physiologic. At least two explanations for this phenomenon are plausible: first, there may be an opening of options for cells after relaxation of a geographical restraint (e.g., when brain cells are injected intravenously) or alternatively, a certain error rate in cell differentiation may occur.
14
Retinal cells arise from multipotential progenitors that may provide a substrate for retinal regeneration. These retinal stem cells, found in low numbers in either the ciliary margin
15 or retina
16 17 give rise to neurons and glia, but few oligodendrocytes in vitro. Although these cells are capable of self-renewal and multilineage differentiation in vitro, they develop mostly into glial cells after engraftment into the adult retina.
18 During vertebrate eye development, the rise of the pigmented epithelium from the proximal optic vesicle is solely dependent on the microphthalmia-inducing transcription factor [MITF].
19 Factors with regulatory function for MITF expression include VIP, which acts also as a differentiation promoter for a functional RPE cell monolayer and has trophic and mitogenic properties in embryonic neural tissue.
20 This 28-amino-acid peptide is found in the aqueous humor, choroid, retina (especially in amacrine cells), and the nerves of the uvea.
21 22 23
The purpose of the present study was to investigate whether ectopic stem cells could be induced to adopt a RPE phenotype. The importance of this approach can be found in age-related macular degeneration (AMD), the leading cause of blindness in developed countries after age 55.
24 The major limiting feature in visual recovery after submacular surgery for exudative AMD is the unavoidable removal of RPE beneath the fovea at the time of surgery. Thus, the rapid repopulation of the bare area of Bruch’s membrane beneath the fovea by RPE is essential for recovery of central vision. Unfortunately, the removed RPE cells are not replaced by the proliferation of adjacent RPE cells. Thus, the simultaneous transplantation of RPE cells before irreversible atrophy of the foveal photoreceptors has occurred is a reasonable option. However, allogeneic adult RPE cells do not attach to senescent Bruch’s membrane efficiently and do not undergo proliferation and spreading to fill in the defect.
25 Furthermore, graft rejection may occur after allogeneic adult RPE transplantation.
26 27 Thus, we would like to study the ability of alternative pluripotent stem cells which have reduced immunogenicity
28 to accomplish this goal more effectively.
Lewis rats, 3 to 4 weeks old, were used in all experiments and were purchased from Harlan Laboratories (Indianapolis, IN). The animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and after approval of the protocol by the University of Louisville Institutional Animal Care and Use Committee (IACUC). A total of 10 eyes, n = 5 per group, were divided into an untreated group and a group injected intravenously with 2% NaIO3 (70 mg/kg; Sigma-Aldrich, St. Louis, MO) through the tail vein. In both groups rNSCs (105 cells/μL) were injected transclerally into the subretinal space of the superior hemisphere of the right eye using a glass micropipette. A microinjector infusion pump was used for injection of a 10-μL cell suspension. A scleral incision was made close to the superior limbus, the micropipette was then inserted into the scleral wound and the fluid was injected into the subretinal space at 1 μL/sec. The treated group received the rNSCs transplant 7 days after the NaIO3 injection.
rNSCs were cultured in proliferating medium without fetal bovine serum (FBS) or in differentiating medium (DMEM/F-12/N2/1%FBS). Increasing concentrations of VIP (5 × 10−9 M, 5 × 10−8 M, 5 × 10−7 M) were used to treat the cells for 5 days and the medium, containing VIP, was changed once. In parallel experiments, the rNSCs were cocultured with RPE at several ratios (1:1; 1:5, or 1:10) or with rat NSR obtained from Lewis rats. In addition, different treatments were combined (e.g., 5 × 10−7 M VIP added to a coculture of RPE cells and rNSCs 1:1). In the coculture experiments, the respective cell types were separated with a membrane (PET [polyethylene terephthalate]; BD Biosciences, Franklin Lakes, NJ) or a cellulose nitrate membrane (Whatman, Maidstone, UK) that allowed only the circulation of soluble factors between the two components.
All experiments were performed three times and results expressed as mean ± SD. Statistical significance (P ≤ 0.05) was determined by Student’s t-test.