Photoreceptor differentiation and integration of retinal progenitor cells transplanted into transgenic rats

Previous studies evaluating neural stem cells transplanted into the mature retina have demonstrated limited levels of graft-host integration and photoreceptor differentiation. The purpose of this investigation is to enhance photoreceptor cell differentiation and integration of retinal progenitor cells (RPC) following subretinal transplantation into retinal degenerate rats by optimization of isolation, expansion, and transplantation procedures. RPCs were isolated from human placental alkaline phosphatase (hPAP)-positive embryonic day 17 (E17) rat retina and expanded in serum-free defined media. RPCs at passage 2 underwent in vitro induction with all trans retinoic acid or were transplanted into the subretinal space of post-natal day (P) 17 S334ter-3 and S334ter-5 transgenic rats. Animals were examined post-operatively by ophthalmoscopy and optical coherence tomography (OCT) at weeks 1 and 4. Differentiation profiles of RPCs, both in vitro and in vivo were analysed microscopically by immunohistochemistry for various retinal cell specific markers. Our results demonstrated that the majority of passage 2 RPCs differentiated into retina-specific neurons expressing rhodopsin after in vitro induction. Following subretinal transplantation, grafted cells formed a multi-layer cellular sheet in the subretinal space in both S334ter-3 and S334ter-5 rats. Prominent retina-specific neuronal differentiation was observed in both rat lines as evidenced by recoverin or rhodopsin staining in 80% of grafted cells. Less than 5% of the grafted cells expressed glial fibrillary acidic protein. Synapsin-1 (label for nerve terminals) positive neural processes were present at the graft-host interface. Expression profiles of the grafted RPCs were similar to those of RPCs induced to differentiate in vitro using all-trans retinoic acid. In contrast to our previous study, grafted RPCs can demonstrate extensive rhodopsin expression, organize into layers, and show some features of apparent integration with the host retina following subretinal transplantation in slow and fast retinal degenerate rats. The similarity of the in vitro and in vivo RPC differentiation profiles suggests that intrinsic signals may have a significant contribution to RPC cell fate determination.     

Growth kinetics and transplantation of human retinal progenitor cells

We studied the growth kinetics of human retinal progenitor cells (hRPCs) isolated from donor tissue of different gestational ages (G.A.), determined whether hRPCs can be differentiated into mature photoreceptors and assessed their ability to integrate with degenerating host retina upon transplantation. Eyes (12-18 weeks G.A.) were obtained with IRB approval and retinas were enzymatically dissociated. Cells were expanded in vitro, counted at isolation and at each passage, and characterized using immunocytochemistry and PCR. GFP positive hRPCs were co-cultured with retinal explants from rd1 and rhodopsin −/− mice, or transplanted into B6 mice with retinal photocoagulation and rhodopsin −/− mice. Eyes were harvested for histological evaluation following transplantation. Our results show that hRPCs from 16 to 18 weeks G.A. had the longest survival in vitro and yielded the maximum number of cells, proliferating over at least 6 passages. These cells expressed the retinal stem cell markers nestin, Ki-67, PAX6 and Lhx2, and stained positively for photoreceptor markers upon differentiation with serum. Some of the GFP positive cells used for transplantation studies showed evidence of migration into the degenerative host retina and expressed rhodopsin. In conclusion, we have determined the growth kinetics of hRPCs and have shown that cells from donor tissue of 16-18 weeks G.A. exhibit the best proliferative dynamics under the specified conditions, and that hRPCs can also be differentiated along the photoreceptor lineage. Further, we have also demonstrated that following transplantation, some of these cells integrate within the host retina and differentiate to express rhodopsin, thereby supporting the potential utility of hRPC transplantation in the setting of retinal degenerative disorders.     

Transplantation of syngeneic Schwann cells to the retina of the rhodopsin knockout (rho(-/-)) mouse

PURPOSE: To determine whether subretinal Schwann cell transplantation can prolong the survival of photoreceptors in the rhodopsin knockout (rho(-/-)) mouse. METHODS: Schwann cells were prepared from postnatal day (PN) 5 to 7 mouse pups and grafted subretinally into the eyes of PN35 rho(-/-) mice. RT-PCR was performed on similarly prepared cells to determine growth factor production in vitro. Eyes were retrieved at PN70 for anatomic and statistical analysis. Control animals received grafts of fibroblasts or sham surgery. RESULTS: RT-PCR demonstrated the presence of message for ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), and glia-derived neurotrophic factor (GDNF) in the cultured Schwann cells. Schwann cell grafts produced a statistically significant rescue of photoreceptors in a restricted area of retina at PN70, but the effect was lost by PN140. Preserved inner segments could be identified, but outer segments were never present. Sham surgery also resulted in photoreceptor rescue but at a reduced level. Fibroblast grafts appeared to produce little or no rescue effect. Grafts of Schwann cells or fibroblasts and sham surgery induced a reactive Müller glial response. CONCLUSIONS: Schwann cells can prolong photoreceptor survival in the rhodopsin knockout mouse until at least PN70.     

Retinal cells integrate into the outer nuclear layer and differentiate into mature photoreceptors after subretinal transplantation into adult mice

Vision impairment caused by degeneration of photoreceptors, termed retinitis pigmentosa, is a debilitating condition with no cure presently available. Cell-based therapeutic approaches represent one treatment option by replacing degenerating or lost photoreceptors. In this study the potential of transplanted primary retinal cells isolated from neonatal mice to integrate into the outer nuclear layer (ONL) of adult mice and to differentiate into mature photoreceptors was evaluated. Retinal cells were isolated from retinas of transgenic mice ubiquitously expressing enhanced green fluorescence protein (EGFP) at either postnatal day (P) 0, P1 or P4 and transplanted into the subretinal space of adult wild-type mice. One week to 11 months post-transplantation experimental retinas were analyzed for integration and differentiation of donor cells. Subsequent to transplantation some postnatal retinal cells integrated into the ONL of the host and differentiated into mature photoreceptors containing inner and outer segments as confirmed by immunohistochemistry and electron microscopy. Notably, the appearance of EGFP-positive photoreceptors was not the result of fusion between donor cells and endogenous photoreceptors. Retinal cells isolated at P4 showed a significant increase in their capacity to integrate into the ONL and to differentiate into mature photoreceptors when compared with cells isolated at P0 or P1. As cell suspensions isolated at P4 are enriched in cells committed towards a rod photoreceptor cell fate it is tempting to speculate that immature photoreceptors may have the highest integration and differentiation potential and thus may present a promising cell type to develop cell replacement strategies for diseases involving rod photoreceptor loss.     

Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light-mediated behavior

PURPOSE: To use progenitor cells isolated from the neural retina for transplantation studies in mice with retinal degeneration. METHODS: Retinal progenitor cells from postnatal day 1 green fluorescent protein-transgenic mice were isolated and characterized. These cells can be expanded greatly in culture and express markers characteristic of neural progenitor cells and/or retinal development. RESULTS: After they were grafted to the degenerating retina of mature mice, a subset of the retinal progenitor cells developed into mature neurons, including presumptive photoreceptors expressing recoverin, rhodopsin, or cone opsin. In rho-/- hosts, there was rescue of cells in the outer nuclear layer (ONL), along with widespread integration of donor cells into the inner retina, and recipient mice showed improved light-mediated behavior compared with control animals. CONCLUSIONS: These findings have implications for the treatment of retinal degeneration, in which neuronal replacement and photoreceptor rescue are major therapeutic goals.     

体外诱导猪Müller细胞定向分化为光感受器细胞的研究

背景 大鼠、小鼠Müller细胞在体外能诱导分化为表达视网膜光感受器细胞特异标记的细胞,但目前有关成年猪Müller细胞分化为视网膜光感受器细胞的研究鲜有报道.目的 研究成年猪Müller细胞于体外定向诱导后分化成视网膜第一级传导神经元光感受器细胞.方法 消化成年猪眼视网膜组织,分离Müller细胞行体外继代单层贴壁培养.使用定向分化培养基对P2、P3和P4代Müller单层贴壁细胞及先形成细胞球悬浮培养2—3d,然后再对贴壁细胞进行诱导分化,最后结合细胞形态和免疫荧光染色结果鉴定Müller细胞以及诱导分化效果.结果 免疫荧光染色结果显示,P2,P3和P4 Müller细胞均能表达Müller细胞特异蛋白分子标记,即谷氨酸合成酶(GS),P3 Müller细胞还同时表达另一种Müller细胞特异蛋白分子标记,神经胶质纤维酸性蛋白(GFAP).免疫荧光染色分析并计算视网膜光感受器细胞特异标记视紫质Rhodopsin阳性率,P2 Müller单层贴壁分化细胞为(27.99±6.53)％,P2悬浮细胞球分化为(16.54±3.40)％.P2悬浮细胞球诱导分化后形态更趋向神经元,而P2代Müller单层贴壁细胞分化后形态仍趋向于成纤维细胞.随着培养代数的增加,细胞球定向分化Rhodopsin阳性表达程度逐渐减弱,P2、P3和P4 Rhodopsin阳性率分别为(56.23±7.32)％、(35.26±8.55)％和(12.68±3.18)％,差异无统计学意义(F=2.618,P=0.099).同代细胞随着诱导时间的延长,Rhodopsin阳性表达有所减弱,差异无统计学意义(P=0.099),但分化细胞形态更为细长.结论 成年猪Müller细胞离体培养后可定向分化为表达视网膜光感受器样细胞特异标记的细胞.细胞球悬浮培养2～3d,以及延长诱导分化时间都能使得分化细胞形态更为细长.     

Revisiting nestin expression in retinal progenitor cells in vitro and after transplantation in vivo

The purpose of this study is to characterize the co-expression of nestin--a neuroectodermal stem cell and a reactive glial marker-with various mature retinal cell markers in retinal progenitor cells (RPCs) expanded in vitro, followed either by in vitro induction or subretinal transplantation. Rat RPCs derived from embryonic day (E) 17 rat retina were expanded in serum free defined culture, and induced to differentiate by all-trans retinoic acid (RA). Following induction, cells were stained for nestin in combination with retinal neuronal and glial markers. Cultured cells were collected for quantitative RT-PCR gene expression analysis prior to and after induction. In a second series, passage 2 RPCs were transplanted into the subretinal space of S334ter-3 retinal degeneration rats at postnatal day 28. After 1-4 weeks, sections through the transplant were double immunostained for nestin and various retinal specific neuronal markers. The cultured RPCs treated with RA exhibited nestin co-expression with various retinal specific markers, including protein kinase C alpha (PKC), neurofilament 200 (NF200), cellular retinaldehyde binding protein (CRALBP), and rhodopsin. Following RA induction, quantitative RT-PCR analysis demonstrated downregulation of nestin, PAX-6, thy1.1, and PKCalpha, and upregulation of rhodopsin, glial fibrillary acidic protein (GFAP), and CrX. No nestin coexpression was observed with any of the retinal specific neuronal markers in RPC transplants in vivo except for some nestin-immunoreactivity overlapping with GFAP positive cells in the host retina. The role of nestin as a unique neural stem/progenitor cell marker should be reconsidered. Nestin expression during RPC maturation appears to be different in vitro versus in vivo.     

Retardation of photoreceptor degeneration in the detached retina of rd1 mouse

PURPOSE: To study the neuroprotective effect of experimental retinal detachment (RD) on photoreceptor degeneration in rd1 mice. METHODS: RD was produced in the eyes of rd1 mice at postnatal day (P) 9. These eyes were collected and compared to controls without RD. The effects of RD on retinal degeneration were evaluated by histochemical staining of nuclei in the outer nuclear layer (ONL), rod and cone photoreceptors, and retinal vessels at P30 in retinal sections and flatmounts. Apoptotic photoreceptors were detected by TdT-mediated dUTP nick-end labeling (TUNEL) at P15. Mice with or without RD were also reared in darkness and evaluated immunohistochemically at P30. RESULTS: The numbers of rhodopsin-positive (rod), peanut agglutinin-positive (cone), and diamino-2-phenyl-indol-stained (rod-plus-cone) cells in the ONL were increased by 2.0-fold, 1.3-fold, and 1.2-fold, respectively, in the rd1 eyes with RD compared to those without RD at P30. In the detached retina, the cone photoreceptor inner/outer segment structures and the deep retinal vessels surrounding the inner nuclear layer and the ONL, but not the ganglion cell layer, were preserved. At P15, TUNEL-positive cell numbers in the ONL were significantly reduced in the eyes with RD. Light exposure had no effect on photoreceptor degeneration in the eyes with or without RD. CONCLUSIONS: RD mediates the preservation of cone and rod photoreceptors in the ONL and surrounding vascular structures by reducing the rate of apoptosis of photoreceptors in rd1 mice. Light deprivation does not appear to be one of the mechanisms of photoreceptor protection in the detached retinas in these mice.     

体外培养人胚胎来源视网膜干细胞的诱导分化

目的 探讨培养的人胚胎来源视网膜干细胞向视网膜终末细胞分化的可能性。方法 来自16～20周人胚胎的视网膜干细胞进行无血清体外培养,并分别进行有血清条件下体外诱导和用含视网膜色素上皮的眼杯模拟体内条件诱导的观察,采用免疫荧光法检测干细胞和视网膜终末细胞表面抗原的表达,采用实时荧光定量PCR法检测诱导前后细胞nestin基因在mRNA水平的表达差异。结果 从人胚胎视网膜神经感觉层分离出的视网膜干细胞,在体外诱导的条件下,可表达视网膜终末细胞标记PKCα、GFAP、Thy1,少数细胞表达nestin和MAP2;在模拟体内环境诱导后,则不仅表达上述细胞标记,而且rhodopsin和syntaxin表达阳性。实时荧光定量PcR法检测显示：诱导后细胞nestin基因表达量较诱导前细胞明显降低。结论 RPE可以促进体外培养的视网膜干细胞向视杆细胞和无长突细胞分化。(中华眼科杂志,2004,40：448-452)     

The role of VEGF and VEGFR2/Flk1 in proliferation of retinal progenitor cells in murine retinal degeneration

PURPOSE: To analyze the role of VEGF and its receptors, VEGFR2/Flk1 and VEGFR1/Flt1, on retinal progenitor cells (RPCs) in a murine model of inherited retinal degeneration (rd1 mice). METHODS: After proliferating RPCs in the retina of rd1 mice were labeled with bromodeoxyuridine (BrdU), expressions of VEGFR2/Flk1 and VEGFR1/Flt1 were immunohistochemically analyzed. To examine its effect on the proliferation of BrdU-positive RPCs in rd1 mice, VEGF was administered into retinal culture medium with or without blocking agents against VEGFR2/Flk1 or VEGFR1/Flt1 in vitro or injected into vitreous cavity in vivo. RESULTS: BrdU-labeled RPCs in rd1 mice expressed VEGFR2/Flk1 but not VEGFR1/Flt1. These cells later expressed retinal neuronal markers such as Pax6 and rhodopsin. Exposure of the retinas from postnatal day (P) 9 rd1 mice to VEGF increased the number of proliferating RPCs by 61% in vitro. This effect was blocked by concomitant administration of VEGFR2/Flk1 kinase inhibitor. In vivo, a single intravitreal injection of VEGF in rd1 mice at P9 increased by 138% the number of RPCs and cells that developed from RPCs in the peripheral retina at P18. CONCLUSIONS: VEGF stimulates the proliferation of RPCs through VEGFR2/Flk1 in rd1 mice. The observed proliferation of RPCs that have the potential to differentiate into retinal neurons may enhance the regeneration of the degenerating retina.     

光感受器移植在视网膜退行性疾病中的应用研究进展

视网膜退行性疾病是影响人类视力的主要疾病，使人感光细胞受损，损害人的视力。而且哺乳动物的视网膜没有再生能力，一旦患病会严重影响人们的正常生活。目前尚未有明确的预防和减缓该类疾病的方法，但移植光感受器以补充视网膜中受损的感光细胞可以治疗视网膜疾病。本文将讨论光感受器移植方法在视网膜退行性疾病中恢复视力的现状和移植技术。     

人视网膜前体细胞的体外视网膜组织片下移植

目的研究人视网膜前体细胞移植到体外培养的人视网膜组织片下的细胞分化。方法取无眼部发育异常的4～5个月胚胎眼球，进行视网膜前体细胞分离培养。将传代的细胞移植到体外培养的视网膜神经上皮组织片下，通过光学显微镜和免疫组织化学观察细胞分化和组织整合情况。结果人视网膜前体细胞在体外培养时形成神经球样细胞团，传代后形成子代细胞团，表达神经干细胞标志Nestin。体外培养的视网膜组织片在5d、10d均能基本维持视网膜结构。移植到视网膜组织片下的视网膜前体细胞能够与其建立细胞连接。这些视网膜前体细胞分化后能够表达胶质纤维酸性蛋白、微管相关蛋白-2和视紫红质，分别为神经胶质细胞、神经元和光感受器细胞的特异蛋白。结论人视网膜前体细胞具有神经干细胞特征，在体外移植到培养的人视网膜组织片下，能够分化成相应的终末分化细胞。     

Gene therapy for detached retina by adeno-associated virus vector expressing glial cell line-derived neurotrophic factor

PURPOSE: To examine the protective effect of glial cell line-derived neurotrophic factor (GDNF) on retinal detachment (RD)-induced photoreceptor damage by using gene delivery. METHODS: Gene delivery to photoreceptors was achieved by subretinal injection of recombinant adeno-associated virus expressing GDNF (rAAV-GDNF) in the right eyes and AAV expressing Escherichia coli LacZ (rAAV-LacZ) in the left eyes of Lewis rats. RD in bilateral eyes was induced with subretinal injection of high-density vitreous substitute in the temporal retina 3 weeks after gene delivery. The synthesis and accumulation of GDNF within the retina was monitored 3 weeks after RD by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), respectively. The rescue of photoreceptors was evaluated by monitoring the preservation of the thickness of photoreceptor outer segment (OS) and outer nuclear layer (ONL). Apoptosis in the photoreceptors was studied using the TdT-dUTP terminal nick-end labeling (TUNEL) method 2 days after RD. Müller cell activity was checked using the immunohistochemistry with glial fibrillary acidic protein (GFAP) antibody 28 days after RD. RESULTS: Gene delivery was demonstrated by immunohistochemical study. The results of ELISA confirmed that high levels of neurotrophic factors were produced in retinas. Photoreceptor OS degeneration and the gradual shortening of the ONL were noted after RD in all the eyes. However, rAAV-GDNF-treated eyes retained longer OS than rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD. ONL was also longer in rAAV-GDNF-treated eyes than in rAAV-LacZ-treated eyes 7 (P = 0.012) and 28 days (P = 0.008) after RD. GDNF-treated eyes had statistically less apoptotic cells than control eyes in photoreceptor layer (P = 0.043). Subretinal proliferation of Müller cells was suppressed in the GDNF-treated group, indicating less scar formation. CONCLUSIONS: GDNF is a potential factor that can protect photoreceptors from degeneration. In addition to preserving the OS and ONL structures, GDNF may exert its protective action by preventing the apoptosis of photoreceptors after RD. GDNF gene therapy may be a valuable adjuvant to current treatments in certain complicated forms of RD.     

人胚眼视网膜光感受器细胞的体外培养和鉴定

目的：探讨人胚眼视网膜光感受器细胞的获得和体外培养方法,为进行人类视网膜光感受器细胞移植提供理论依据。方法：采用酶分层消化法分离获取了胚眼纯视网膜光感受器细胞,使用视网膜色素上细胞的条件培养液,在体外长期培养并进行形态学鉴别;用苏木素－伊红染色显示消化后的视网膜组织结构层次,以确定所获得取的视网膜光感受器细胞的纯度;用抗视杆细胞特异性视紫质蛋白抗体（rhodopsin4D2,Rho4D2)免疫细胞化学染色做细胞鉴定,结果：酶分层消化法能获取较纯的视网膜光感受器细胞,抗Rho4D2阳性,并能在体外存活较长时间,结论：酶分层消化法所获取的人胚眼纯视网膜光感受感器细胞能在体外较长时间存活,并可表达光感受器细胞特异性视紫质蛋白,将为人类视网膜光感受器细胞的移植和各种体外研究提供细胞特异性视紫质蛋白,将为人类视网膜光感受器细胞的移植和各种体外研究提供细胞来源。     

Current approaches and future prospects for stem cell rescue and regeneration of the retina and optic nerve

The 3 most common causes of visual impairment and legal blindness in developed countries (age-related macular degeneration, glaucoma, and diabetic retinopathy) share 1 end point: the loss of neural cells of the eye. Although recent treatment advances can slow down the progression of these conditions, many individuals still suffer irreversible loss of vision. Research is aimed at developing new treatment strategies to rescue damaged photoreceptors and retinal ganglion cells (RGC) and to replace lost cells by transplant. The neuroprotective and regenerative potential of stem and progenitor cells from a variety of sources has been explored in models of retinal disease and ganglion cell loss. Continuous intraocular delivery of neurotrophic factors via stem cells (SC) slows down photoreceptor cells and RGC loss in experimental models. Following intraocular transplantation, SC are capable of expressing proteins and of developing a morphology characteristic of photoreceptors or RGC. Recently, recovery of vision has been achieved for the first time in a rodent model of retinal dystrophy, using embryonic SC differentiated into photoreceptors prior to transplant. This indicates that clinically significant synapse formation and acquisition of the functional properties of retinal neurons, and restoration of vision, are distinct future possibilities.     

Cell cycle regulation in retinal progenitors by glia-derived neurotrophic factor and docosahexaenoic acid

PURPOSE: A recent study has shown that glia-derived neurotrophic factor (GDNF) and docosahexaenoic acid (DHA) promote the survival and differentiation of retina photoreceptors. The current study was undertaken to investigate whether these molecules participate in cell cycle regulation in retinal progenitors in vitro. METHODS: Developmental changes in the expression of the stem cell marker nestin and of cell cycle and differentiated neuron markers were analyzed in neuroblasts obtained from 1-day-old rat retinas. The effects of GDNF and DHA on those changes were then determined. RESULTS: Expression of nestin, found in more than one third of neuroblasts at day 1, rapidly decreased during development, with most neuroblasts acquiring the photoreceptor phenotype. GDNF increased the percentage of photoreceptor progenitors expressing nestin, whereas DHA reduced it, simultaneously enhancing photoreceptor differentiation. Several markers of cell cycle progression indicated that photoreceptor progenitors maintained an active cell cycle during the first 2 days in vitro. GDNF stimulated the cell cycle, increasing the number of dividing cells and generating more photoreceptor progenitors, whereas DHA induced cell cycle exit and photoreceptor differentiation. Analysis of the expression of the cyclin-Cdk inhibitor p27(Kip1) confirmed these results. CONCLUSIONS: GDNF and DHA acted as molecular cues, counterbalancing the decision of photoreceptors to remain in or exit the cell cycle. The results strongly suggest that both factors participate in determining the number of photoreceptors in vitro, regulating the cell cycle and survival at early and late stages of development, respectively. Hence, GDNF and DHA may coordinately control the histogenesis of photoreceptors in the retina by modulating both neurogenesis and apoptosis.     

A role for the Tubby-like protein 1 in rhodopsin transport

PURPOSE: To test the hypothesis that a lack of Tubby-like protein 1 (TULP1) function causes aberrant transport of nascent rhodopsin and to examine the functional relationship between the homologous proteins TULP1 and Tubby by studying mice carrying combined mutations. METHODS: Subcellular localization of TULP1 and rhodopsin in photoreceptors was determined by immunofluorescence and by postembedding immunoelectron microscopy. Mice carrying different tulp1/tubby allele combinations were examined by histology, electroretinograms (ERGs), and immunofluorescence microscopy. RESULTS: TULP1 is distributed throughout the photoreceptor cytoplasm but is excluded from the outer segments and the nuclei. In the tulp1-/- mice, ectopic accumulation of rhodopsin occurs at an early age. Both the vesicular profiles in the interphotoreceptor space and the inner segment plasma membranes are immunoreactive for rhodopsin. Mice doubly homozygous for null mutations in the tulp1 and tubby genes initially develop photoreceptors and express a battery of photoreceptor markers at age 14 days. Thereafter their photoreceptors undergo a fulminant degeneration that reaches completion by postnatal day 17. The disease phenotype in the double homozygote is much more severe than either single homozygote. Double heterozygotes are phenotypically normal. CONCLUSIONS: A lack of TULP1 function results in misrouting of nascent rhodopsin. TULP1 may be a component of the cellular machinery that targets nascent rhodopsin to the outer segments. Comparison of disease phenotypes in the single and double mutants suggests that TULP1 and Tubby are not functionally interchangeable in photoreceptors nor do they form an obligate functional complex.     

Retinal cAMP levels during the progression of retinal degeneration in rhodopsin P23H and S334ter transgenic rats

PURPOSE: To test whether high levels of cAMP promote apoptosis and shorten the life of retinal rod photoreceptors, the changes in cAMP levels during retinal degeneration were analyzed in two transgenic rat models that express rhodopsin P23H and S334ter mutations. METHODS: Dark- and light-adapted heterozygous P23H (lines 1 and 3; P23H-1 and -3), S334ter line 4 (S334ter-4), and Sprague-Dawley (control) rats were studied at 4 to 8 weeks by cAMP enzyme competitive immunoassay and by cAMP immunocytochemistry. RESULTS: In control animals retinal cAMP content reached a steady state level at 30 days of age. Dark-adapted control retinas had up to 97% higher cAMP content than light-adapted retinas, and photoreceptor cells were the major source of this increase. Dark-adapted photoreceptors in all three lines of transgenic rats at advanced stages of retinal degeneration had cAMP content different from that of the control. In rats that express mutant rhodopsin, the number of photoreceptor cells was progressively reduced, because of retinal degeneration, but dark-adapted cAMP levels did not decline accordingly. P23H transgenic animals of both lines had higher levels of cAMP per photoreceptor cell count than control animals. This elevation was more pronounced as degeneration progressed. S334ter animals showed smaller cAMP elevation than P23H rats at a similar stage of retinal degeneration, but at a point when S334ter rats were undergoing rapid retinal degeneration, whereas in P23H rats retinal degeneration was slowing down. CONCLUSIONS: All three lines of transgenic rats carrying rhodopsin mutations show an increase in dark-adapted photoreceptor cAMP levels. A complex relationship exists between cAMP levels and the rate of cell death in the retina. Although initially higher levels of cAMP may promote cell survival and slow down retinal degeneration, ultimately, elevated cAMP levels may become toxic and may contribute to retinal cell death.     

Transplantation of full-thickness retina in the rhodopsin transgenic pig

PURPOSE: To establish the morphology of full-thickness neuroretinal grafts transplanted to hosts with degenerative photoreceptor disease. METHODS: Twenty rhodopsin transgenic pigs received a neuroretinal sheet from a neonatal normal pig in one eye. Following vitrectomy and retinotomy with bleb formation, the grafts were positioned inside the bleb between the host neuroretina and retinal pigment epithelium. After a survival time of 4 months, eye specimens were studied by light and electron microscopy as well as with immunohistochemical markers. RESULTS: One eye developed endophthalmitis in the immediate postoperative period and was terminated. Laminated grafts with correct polarity were found in 13 of the remaining 19 eyes. In most cases, these grafts had well-developed organized photoreceptors with outer segments apposed to the host retinal pigment epithelium. The inner layers of the graft contained mostly Müller cells. Both eyes of the hosts had a reduction of photoreceptor cells in most of the retina, while inner layers remained relatively intact. CONCLUSIONS: Full-thickness neuroretinal grafts can be transplanted to a large animal host with photoreceptor degeneration. The transplantation procedure is relatively atraumatic to both graft and host tissue, and the grafts survive well for at least 4 months. The graft and host retina does not seem to form extensive neuronal contacts, and future work must be directed at stimulating such activity without disrupting the retinal neuronal organization.     

Transplantation of rat embryonic stem cell-derived retinal cells restores visual function in the Royal College of Surgeons rats

Aim of study

To evaluate the feasibility of transplantation of embryonic stem cell (ESC)-derived retinal cells in the treatment of retinal degeneration.

Materials and methods

Rat ESCs were isolated and induced into retinal progenitor cells (RPCs) in vitro, which were subsequently induced into retinal pigment epithelium cells (RPEs) and photoreceptors (PRCs). All cells were identified by Western blot detection of their specific markers. RPEs and PRCs were, respectively, injected into the retina of Royal College of Surgeons (RCSs) rats. Control group was injected with PBS. Post-transplantation visual function was determined by electroretinography (ERG). The histology of the whole eye was compared by H&E staining.

Results

RPEs and PRCs were successfully derived from rat ESCs through the two-step differentiation as indicated by the presence of ESC- (Oct-3/4, Nanog, TRA-1-60 and TRA-1-81), RPC- (Rx, Mitf, Pax6 and Chx10), RPE- (RPE65 and keratin) and PRC-specific markers (blue opsin, red/green opsin, recoverin and rhodopsin) in Western blot. The amplitude of ERG a- and b-wave in RPE- and PRC-transplanted groups at week 2 and 10 after transplantation was markedly higher compared with PBS controls. Retinal injury and vascular stress response was not detected in any of the RCS rats after transplantation.

Conclusion

The developed stepwise protocol can derive retinal cells from ESCs. Transplantation of these retinal cells can restore visual function of RCS rats. Our study provides evidence for potential clinical application of ESC-based cell therapy for retinal degeneration.