Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

In mammals, multipotent stem cells are generally more restricted in their developmental capacity and are largely programmed to develop into certain cell types within a specific tissue or organ. However, recent evidence suggests that these stem cells also have the capacity to differentiate into cell types specific for the tissue into which they are transplanted. For example, neural stem cells (NSCs) have been shown to differentiate into bone marrow derived cells when grafted into the bloodstream of irradiated hosts (36). Conversely, bone marrow-derived stem cells have been shown to differentiate into retinal cells when transplanted into injured rat retina (37). This capability to display more potential phenotypes in alternate niches could potentially allow stem cells derived from one body part to form cells of other body parts, a quality called plasticity. This experimental data remains controversial and it will likely be many years before the true plasticity of stem cells is fully understood.

Over the last few years, multipotent stem cells have been isolated from different parts of the central nervous system (CNS) including the brain, retina, and spinal cord. These “CNS stem cells” include NSCs that are brain derived and have the capability to generate all cells of the CNS lineage, namely neurons, astrocytes, and oligodendrocytes, as well as RSCs that can generate retinal-specific neurons, but not oligodendrocytes, as none are normally found within the retina. The isolation of stem/progenitor cells from various regions of the CNS has raised the possibility of using them as a donor cell source for retinal transplantation, where they offer great promise for repair of the diseased retina.

The mammalian retina presents formidable challenges to medical therapeutics because of its restricted capacity for endogenous self repair and regeneration. In addition, attempts at exogenous repair/restoration are severely constrained by its delicate structure and cytoarchitectural complexity. The lack of retinal regeneration and the paucity of effective strategies for repair invariably result in partial or complete visual deficits in patients suffering from retinal degenerations. Tissue engineering using progenitor cells isolated from the CNS offers a potential therapy for retinal degenerations because CNS stem cells are not only multipotent and capable of self-renewal, but also, more importantly, they satisfy the immunogenic requirement for CNS transplantation by having an intrinsic immune privilege status, as will be discussed shortly.

RETINAL TRANSPLANTATION OF CNS STEM CELLS

A major technical challenge facing attempts at intraocular grafting of fetal retina is not so much survival or differentiation, but instead a lack of widespread functional integration of the graft with the remaining circuitry of the host retina. Thus, a fundamental prerequisite to functional success is widespread integration of graft-derived neurons within the mature degenerating mammalian retina. Furthermore, the grafted cells must exhibit this capacity in the face of active retinal disease.

These criteria were first met by hippocampal progenitor cells isolated and grown from the brain of adult rats (38). Hippocampal progenitor cells are a type of NSC derived from a region of active neurogenesis in adult mammals. In the first report on grafting this type of cell to the retina, Takahashi et al. demonstrated that intravitreal injections of hippocampal progenitors resulted in a spectacular degree of morphological integration within the neural retina of neonatal rats (39). In a subsequent article, we reported that these rat hippocampal progenitor cells were capable of widespread migration and

Fig. 2. Localization of grafted adult hippocampal progenitor cells (AHPCs) to specific retinal layers. Cells were grafted into the vitreous of 4 (A–D), 10 (E), and 18 (F) -wk-old rats, and examined 4 wk later. Sections were stained with antisynaptophysin/Cy3 antibody and viewed under flouroscein isothyocyanate (FITC) and Cy3 fluorescent illumination. Arrow in A indicates cell seen in B at higher power; arrow in C indicates cell seen in D at higher power. vit, vitreous; gcl, ganglion cell layer; ipl, inner plexiform layer; inl, inner nuclear layer; opl, outer plexiform layer; onl, outer nuclear layer; srs, subretinal debris and degenerating photoreceptor elements. (Reprinted from ref. 46, with permission.)

morphological integration into the degenerating retina of mature Royal College of Surgeons rats during the phase of active retinal degeneration (40). In this case, grafted cells differentiated into neurons based on marker expression and morphologically showed indications of an ability to respond to the host retinal cytoarchitecture (Fig. 2). For instance, the somata of grafted cells were located predominantly within the cellular layers of the retina, whereas their processes extended into the plexiform layers, frequently at an appropriate orientation, and with finer processes branching off within

specific sublaminae (Fig. 2). In addition, GFP+ donor cells within the ganglion cell layer expressed the neuronal marker NF-200, known to be expressed by retinal ganglion cells, and extended large numbers of growth cone-tipped processes into the host optic nerve. We have also found that xenografts of neural progenitors can integrate with the host retina, although there may well be limits to this capacity (41).

Indeed, the factors controlling the integration of stem and progenitor cells within the CNS are likely to be many. In the case of xenografts, basic metabolic and size considerations will necessarily play a role. In addition, the degree of genetic disparity between graft and host will, to some extent, limit the efficacy of intercellular signaling. Furthermore, the relative plasticity of the donor cell line will influence outcome, as will the developmental state of the host and any ongoing degeneration or inflammation. Finally, there is the important factor of immune tolerance. Recent studies provide a baseline for predicting the immunological consequences of transplanting stem and progenitor cells to various sites within the CNS, as will be discussed after further considering the relationship between stem cell plasticity and graft-host integration.

Plasticity vs Commitment: A Stem Cell Conundrum

Plasticity, the very property that endows stem cells with the ability to engraft in the mature CNS, also carries with it a significant burden. The more plasticity a cell possesses, the less committed it is to a specific lineage. Although the lack of commitment of an embryonic stem (ES) cell can be exploited to generate a number of cell types from a single cell source, it is also clear that ES cells cannot, at least at present, be induced to differentiate into all types of cells. In those cell types that can be generated, it remains unclear how “complete” the differentiation truly is, i.e., it may look like a specific cell, or even express markers of that cell, but does it become a fully mature and functional replica of that cell? The dichotomy between plasticity and commitment is perhaps the most important issue to be addressed in the field of stem cell biology. This relationship is especially true in the context of retinal transplantation.

Both embryonic and mature retinal tissue has been used for transplantation studies in animals and humans for decades. Although these grafts invariably differentiate into mature retinal tissue, they have shown a very limited capacity to integrate (e.g., migrate or send neurites into the host retina). Our early work with hippocampal stem cells showed the opposite result: engraftment, but only limited differentiation into retinal cell types. We further investigated this issue in a controlled study by grafting neural stem cells, or developing retina, into the same hosts (rd mice). Our results reinforced the hypothesis that plasticity and commitment are in some ways mutually exclusive. Conventional tissue grafts have the intrinsic potential to differentiate into the lineages from which they were obtained, but lack the plasticity to fully engraft in mature hosts. Conversely, stem cells have the intrinsic ability to engraft, but often differentiate incompletely or in unpredictable ways. At present, we do not possess the tools to take the reins of stem cell development and induce them to make the cell types we need or desire. We describe this in terms of the experimenter lacking the tools, rather than an intrinsic lack of developmental potential of the stem cell, because it is clear that ES cells, for example, have the “potential” to make all cells in the body. Harnessing the power of stem

cells for therapeutic purposes requires that we unravel the secrets of development, thereby unleashing the potential for stem cells to repair the diseased or injured body.

Immunological Aspects of Stem Cell Transplantation

A series of in vitro and in vivo experiments have revealed mouse NPCs to be immune privileged cells. These cells survive allografting without the need for immune suppression and are more likely to be immunologically tolerated than solid tissue grafts of brain or retina, which contain major histocompatibility complex (MHC) class II-expressing microglia. On a cautionary note, the immunological situation in large mammals, including humans, may be more complex than in the mouse. For instance, we already know that human NPCs express abundant MHC class I, although, like their murine counterparts, they do not express class II (42). Because NPCs appear to exhibit relative immune privilege as a donor cell type, and because transplantation of these cells is directed towards recipient cites that are themselves immune privileged, such as the retina, brain, or spinal cord, it seems reasonable to conclude that transplantation of these cells will prove to be less challenging from an immunological perspective than has been the case with hematopoietic stem cells (that express class II MHC antigens) or solid organ transplants (that contain class II-expressing passenger leukocytes). It can be hoped that systemic immunosuppression will not be necessary when grafting NPCs to the retina clinically, although it remains to be seen whether this is in fact the case.

Transplantation of Retinal Stem Cells

A profound degree of engraftment can be achieved through the use of brain-derived progenitor cells for retinal transplantation. It has become apparent, however, that brainderived cells do not differentiate into authentic retinal neurons in the microenvironment of the mature, diseased retina. Several possible strategies can be employed in an effort to overcome this obstacle. For example, one could either attempt to modify a brainderived cell to induce transdifferentiation along a retinal lineage, or induce the differentiation of a more plastic, less differentiated cell type such as an ES cell into such a fate. We and several other groups have chosen a third strategy, namely, the isolation of progenitor cells from the neural retina of the developing mammalian eye (Fig. 3) (34). Although these studies are in the early stages, one important discovery is that these cells, upon transplantation to the retina of adult mammals with retinal disease, possess both the integrative plasticity that is a hallmark of CNS stem cells, as well as the ability to differentiate into retinal neurons, including photoreceptors (Fig. 4). We are hopeful that further studies of retinal progenitor cell grafts will point the way forward to the development of clinical strategies aimed at restoring vision to the blinded eye.

Functional Repair of the Diseased Retina

The results of the previous studies make a strong case for the need for RSCs. If one could isolate stem or progenitor cells from the retina, the properties of self renewal and multipotentiality could allow for a large supply of donor cells for use in retinal repair. Researchers have identified and isolated RSCs from both the mature ciliary marginal zone and the developing neurosensory retina.

The ciliary marginal zone stem cells have an impressive ability to replicate in the absence of proteins and have been shown to differentiate into cells expressing retinal markers in culture. On a cautionary note, however, that same lack of growth factor dependence, together with limited differentiation potential in the mature, diseased retina, makes them a questionable choice for transplantation studies.

The work of Ahmad’s group has provided a wealth of information on the intracellular signaling pathways of RSCs derived from developing retinal tissue. We have also been studying a variety of sources for isolation of RSCs. Neural retina from the period of late neurogenesis (postnatal day 1 [P1] for mice) appears to be optimal for generating cells that both expand through repeated passaging in culture and generate retinal cells upon transplantation to the mature host.

We recently described the utilization of this source of RSCs in detail (34). In culture, we demonstrated that RPCs isolated from P1 GFP mice could be greatly expanded while maintaining their multipotentiality, which included the expression of rod, bipolar, and glial markers by distinct subsets of differentiating cells. Under proliferation conditions, RPCs expressed a number of neurodevelopmental genes and surface markers. Reverse transcriptase-polymerase chain reaction demonstrated expression of nestin and Sox2, as well as other neurodevelopmental genes including Notch1, Hes1, Hes5, Sox2, Prox1, Mash1, numb, and NeuroD. Analysis by flow cytometry showed surface expression of GD2 ganglioside, CD15 (LeX), and the tetraspanins CD9 and CD81.

After grafting to the degenerating retina of mature mice, a subset of the retinal progenitor cells developed into mature neurons, including cells expressing the photoreceptor markers recoverin, rhodopsin, or cone opsin. Importantly, cells could be observed differentiating into photoreceptors that had both morphological and cytochemical hallmarks of mature rods. When grafted into rho–/– hosts, we found rescue of host cells in the outer nuclear layer (ONL), along with widespread integration of donor cells into the inner retina. A subset of grafted cells expressed cone markers in this model. Moreover, recipient mice showed improved light-mediated behavior compared to controls. Greater thickness of the host ONL was seen in stem cell-grafted eyes, but not in sham-operated controls and photoreceptor density was higher in the treated eye than the untreated eye. The increase in photoreceptor density correlated with graft location. Graft-associated rescue likely reflects an indirect neuroprotective effect, similar to that reported previously (9,42,44). The behavioral results suggest that grafted RPCs decrease the tempo of luminance detection loss in dystrophic (rho–/–) mice, especially at low light levels. Preservation of visual function was detected over a 25-wk period, extending into a period with limited photoreceptor survival. As there are a number of technical difficulties associated with functional assessment in mice, expanding this approach to larger animals would be useful for accurate determination of graft efficacy, as well as development of surgical approaches for potential clinical application of transplantation research.

Human Retinal Progenitor Cells

RPCs can also be obtained from cadaveric human retinal tissue. We have recently reported that retinas obtained from postmortem premature infants can be enzymatically dissociated, and viable proliferative cells obtained and grown in the presence of epithelial growth factor and basic fibroblast growth factor. Such cultures grow to confluence

repeatedly for up to 3 mo. Again, the cells can be grown as suspended spheres or adherent monolayers, depending on how they are cultured. Cultured human RPCs (hRPCs) express a range of markers consistent with CNS progenitor cells and similar to those found in human BPCs (hBPCs) from the same donors, including nestin, nucleostemin, vimentin, Sox2, and the proliferation marker Ki-67. Also expressed are the surface markers GD2 ganglioside, CD15 (Lewis X), the tetraspanins CD9 and CD81, the CD95 “death receptor” (Fas), CD133, and MHC class I antigens, however, no MHC class II expression was detected, nor was expression of Pou5f1 (Oct4) or Nanog (Schwartz and Klassen, unpublished data), two genes expressed in ES cells. hRPCs, but not hBPCs, expressed the genes for Dach1, Pax6, Six3, Six6, and recoverin. hBPCs, but not hRPCs, expressed the genes for Dlx2, Dlx5, Gad67, and Olig2 (Schwartz and Klassen, unpublished data). Minority subpopulations of both hRPCs and hBPCs expressed the protoneuronal genes doublecortin and β-III tubulin, as well as the glial gene GFAP (glial fibrillary acid protein), consistent with increased lineage restriction in subsets of cultured cells. These data suggest that although immature neuroepithelial cells taken from different regions of the immature CNS express a number of gene products common to CNS progenitor cells, they also retain genes indicative of fate specification events occurring in the region from which they were harvested.

Based on these findings, it is evident that hRPCs, derived from retinal tissue obtained postmortem from premature infants at just past mid-gestation, represent a rather heterogeneous population of progenitors. Estimating the neurodevelopmental age of the 7-d-old rodent to be roughly equivalent to the newborn human, a mid-gestation human approximately corresponds to the E13 rodent. Studies of the E13 rodent retina have shown that it contains precursors of rods, amacrine cells, cones, ganglion cells, and horizontal cells, with little evidence of bipolar or Müller cell development (45). Our immunocytochemical studies of progenitors from the developing human retina are entirely consistent with a heterogeneous population, based on morphology and marker expression, suggesting that in vitro cultures represent to some extent the heterogeneity found in vivo. The presence of subpopulations expressing doublecortin (DCX), recoverin, β-III tubulin, and GFAP (Fig. 5) among the majority of cells expressing the immature markers nestin, Sox2, and vimentin, suggests a tendency toward continuous differentiation in these cultures, even under proliferation conditions (Fig. 5). DCXand GFAP-expressing subpopulations were also present in brain-derived human progenitors, however, further studies will be necessary to determine whether these findings are specific to cells harvested at this particular developmental time point or can be generalized to a wider range of CNS progenitors, either human or from other species. Another point of considerable interest is the potential of hRPCs to integrate and differentiate into retinal neurons following transplantation, particularly in the setting of photoreceptor loss.

CONCLUSIONS

Stem cell biology is providing new insight into the development and pathophysiology of the mammalian retina. Retinal specificity of cell fate is now known to relate to changes in transcription factor expression during lineage choices made by stem and progenitor cell populations. Furthermore, the responses of grafted stem cells to retinal

Fig. 5. Phenotypic markers in human retinal progenitor cell cultures. A–E = proliferation conditions; F = differentiation conditions. (A) Nestin (green) staining showed a cytoplasmic pattern, Sox2 (red) a nuclear pattern, and GD2 ganglioside (blue) a surface pattern consisting of discrete punctate foci of variable size and number. (B) CD15 immunoreactivity (red) was variable and most evident on a subset of cultured cells with small, rounded profiles (shown against phase constrast). (C) Distinct subpopulations within hRPC cultures expressed either the neuronal marker b-III tubulin (red) or the glial marker GFAP (green). (D) Other subpopulations could be distinguished by expression of the neuroblast marker DCX (red) as compared to the photoreceptor marker recoverin (green). (E) Nestin (blue) and Sox2 (red) co-localized with Ki-67 (green) in hRPCs grown under proliferation conditions. (F) Under proneuronal differentiation conditions, there was an absence of Ki-67 staining, whereas Sox2 (red) was still detectable but now assumed a perinuclear distribution together with cytoplasmic expression of FRRI (blue). Original magnification X40, except F = X100. Bars = 50 m, except E,F = 10 m. (Reprinted with permission from ref. 47, with permission.)

injury cues, such as photoreceptor degeneration, provide a means of evaluating local homeostatic mechanisms in the diseased microenvironment. Although much work remains to be done, especially with respect to the investigation of host visual benefits after transplantation, it is already apparent that RSC transplantation provides an important new strategy for altering the course of retinal degenerations.

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