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.     

Loss of photoreceptor potential from retinal progenitor cell cultures, despite improvements in survival

Retinal degeneration (RD) results from photoreceptor apoptosis. Cell transplantation, one potential therapeutic approach, requires expandable stem cells that can form mature photoreceptors when differentiated. Freshly dissociated primary retinal cells from postnatal day 2-6 (PN2-6) mouse retina can give rise, post-transplantation, to photoreceptors in adult recipients. Unfortunately, incorporation rates are low; moreover, photoreceptor potential is lost if the same PN2-6 cells are cultured prior to transplantation. We investigated the identity of the cells forming photoreceptors post-transplantation, using FACS sorted primary postnatal day (PN) 3-5 Rho-eGFP retinal cells. Higher integration rates were achieved for cells that were expressing Rho-eGFP at PN3-5, indicating that post-mitotic photoreceptor precursors already expressing rhodopsin form the majority of integrating rods. We then investigated improvement of cell culture protocols for retinal progenitor cells (RPCs) derived from PN3-5 retinal cells in vitro. We succeeded in improving RPC survival and growth rates 25-fold, by modifying retinal dissociation, replacing N2 supplement with B27 supplement minus retinoic acid (B27 − RA) and coating flasks with fibronectin. However, levels of rhodopsin and similar photoreceptor-specific markers still diminished rapidly during growth in vitro, and did not re-appear after in vitro differentiation. Similarly, transplanted RPCs, whether proliferating or differentiated, did not form photoreceptors in vivo. Cultured RPCs upregulate genes such as Sox2 and nestin, markers of more primitive neural stem cells. Use of these cells for RD treatment will require identification of triggers that favour terminal photoreceptor differentiation and survival in vitro prior to transplantation.     

BDNF-treated retinal progenitor sheets transplanted to degenerate rats: improved restoration of visual function

The aim of this study was to evaluate the functional efficacy of retinal progenitor cell (RPC) containing sheets with BDNF microspheres following subretinal transplantation in a rat model of retinal degeneration. Sheets of E19 RPCs derived from human placental alkaline phosphatase (hPAP) expressing transgenic rats were coated with poly-lactide-co-glycolide (PLGA) microspheres containing brain-derived neurotrophic factor (BDNF) and transplanted into the subretinal space of S334ter line 3 rhodopsin retinal degenerate rats. Controls received transplants without BDNF or BDNF microspheres alone. Visual function was monitored using optokinetic head-tracking behavior. Visually evoked responses to varying light intensities were recorded from the superior colliculus (SC) by electrophysiology at 60days after surgery. Frozen sections were studied by immunohistochemistry for photoreceptor and synaptic markers. Visual head tracking was significantly improved in rats that received BDNF-coated RPC sheets. Relatively more BDNF-treated transplanted rats (80%) compared to non-BDNF transplants (57%) responded to a "low light" intensity of 1cd/m2 in a confined SC area. With bright light, the onset latency of SC responses was restored to a nearly normal level in BDNF-treated transplants. No significant improvement was observed in the BDNF-only and no surgery transgenic control rats. The bipolar synaptic markers mGluR6 and PSD-95 showed normal distribution in transplants and abnormal distribution of the host retina, both with or without BDNF treatment. Red-green cones were significantly reduced in the host retina overlying the transplant in the BDNF-treated group. In summary, BDNF coating improved the functional efficacy of RPC grafts. The mechanism of the BDNF effects--either promoting functional integration between the transplant and the host retina and/or synergistic action with other putative humoral factors released by the RPCs--still needs to be elucidated.     

The relationship of photoreceptor degeneration to retinal vascular development and loss in mutant rhodopsin transgenic and RCS rats

The early loss of photoreceptors in some retinal degenerations in mice has been shown to have a profound effect on vascular development of the retina. To better characterize this relationship, we have examined the formation of retinal blood vessels during the first month of life in 8 lines of transgenic rats with different ages of onset and rates of photoreceptor cell loss mediated by the expression of mutant rhodopsin (P23H and S334ter). The number of capillary profiles in the superficial plexus (SP) and deep capillary plexus (DCP) of the retina were quantified in retinal sections taken at postnatal day (P) 8, 10, 12, 15 and 30. In normal wild-type rats, the SP and DCP had mostly established mature, adult patterns by P15, as previously shown. In the transgenic rats, the loss of photoreceptors had relatively little effect on the SP. By contrast, the loss of photoreceptors during vascular development had a major impact on the DCP. In the two lines with early and most rapid photoreceptor loss, S334ter-7 and S334ter-3, where about 90% and 65%, respectively, of the photoreceptors were already lost by P15, the DCP either failed to form (S334ter-7) or the number of capillary profiles was less than 7% of controls (S334ter-3). In lines where almost all photoreceptors were still present at P15 (S334ter-4, S334ter-9, P23H-2 and P23H-3), the number of profiles in the DCP were the same as in wild-type controls at P30. In two lines with an intermediate rate of degeneration (S334ter-5 and P23H-1), where only about 25% of the photoreceptors were lost by P15, there was an intermediate number of vascular profiles in the DCP at P30. Thus, a very close relationship between the number of photoreceptors and vessel profiles in the DCP during its development exists in the transgenic rats, and the loss of photoreceptors results in the failure or inhibition of the DCP to develop. Several mechanisms may explain this relationship including changes in the level of physiological oxygen tension or alteration in the release of angiogenic factors that normally drive vessel development. Analysis of older transgenic retinas up to 1 year of age revealed that (1) vascular profiles are lost from the DCP in essentially all lines once fewer than about 30–33% of photoreceptors remain; (2) in those lines where the DCP essentially did not develop (S334ter-7 and S334ter-3), the effect of photoreceptor absence was permanent, and there was no late vascularization of the DCP; (3) the number of capillary profiles in the SP remained no different from controls in any of the lines, despite long-standing loss of photoreceptors; and (4) neovascularization of the RPE by retinal capillaries occurred with a latency of 60–180 days after the loss of photoreceptors, except in S334ter-7 rats, where neovascularization essentially did not occur. Analysis of RCS rats was carried out for comparison.     

The relationship of photoreceptor degeneration to retinal vascular development and loss in mutant rhodopsin transgenic and RCS rats

The early loss of photoreceptors in some retinal degenerations in mice has been shown to have a profound effect on vascular development of the retina. To better characterize this relationship, we have examined the formation of retinal blood vessels during the first month of life in 8 lines of transgenic rats with different ages of onset and rates of photoreceptor cell loss mediated by the expression of mutant rhodopsin (P23H and S334ter). The number of capillary profiles in the superficial plexus (SP) and deep capillary plexus (DCP) of the retina were quantified in retinal sections taken at postnatal day (P) 8, 10, 12, 15 and 30. In normal wild-type rats, the SP and DCP had mostly established mature, adult patterns by P15, as previously shown. In the transgenic rats, the loss of photoreceptors had relatively little effect on the SP. By contrast, the loss of photoreceptors during vascular development had a major impact on the DCP. In the two lines with early and most rapid photoreceptor loss, S334ter-7 and S334ter-3, where about 90% and 65%, respectively, of the photoreceptors were already lost by P15, the DCP either failed to form (S334ter-7) or the number of capillary profiles was less than 7% of controls (S334ter-3). In lines where almost all photoreceptors were still present at P15 (S334ter-4, S334ter-9, P23H-2 and P23H-3), the number of profiles in the DCP were the same as in wild-type controls at P30. In two lines with an intermediate rate of degeneration (S334ter-5 and P23H-1), where only about 25% of the photoreceptors were lost by P15, there was an intermediate number of vascular profiles in the DCP at P30. Thus, a very close relationship between the number of photoreceptors and vessel profiles in the DCP during its development exists in the transgenic rats, and the loss of photoreceptors results in the failure or inhibition of the DCP to develop. Several mechanisms may explain this relationship including changes in the level of physiological oxygen tension or alteration in the release of angiogenic factors that normally drive vessel development. Analysis of older transgenic retinas up to 1 year of age revealed that (1) vascular profiles are lost from the DCP in essentially all lines once fewer than about 30-33% of photoreceptors remain; (2) in those lines where the DCP essentially did not develop (S334ter-7 and S334ter-3), the effect of photoreceptor absence was permanent, and there was no late vascularization of the DCP; (3) the number of capillary profiles in the SP remained no different from controls in any of the lines, despite long-standing loss of photoreceptors; and (4) neovascularization of the RPE by retinal capillaries occurred with a latency of 60-180 days after the loss of photoreceptors, except in S334ter-7 rats, where neovascularization essentially did not occur. Analysis of RCS rats was carried out for comparison.     

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.     

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。     

Retinal transplantation-induced recovery of retinotectal visual function in a rodent model of retinitis pigmentosa

PURPOSE: To map the spatiotemporal decline in retinally driven activity in the superior colliculus (SC) of transgenic S334ter-line-3 rats that express a mutated rhodopsin, which causes photoreceptor degeneration. To determine whether transplantation of fetal retinal sheets into the subretinal space of these rats can recover visual activity in the SC. METHODS: A visual stimulus was presented to the eye, and responses were recorded across the SC of untreated S334ter-line-3 rats aged 28 to 288 days. These data were used to draw a map of the developing scotoma. Intact retinal sheets from embryonic day 19 rats were transplanted into the subretinal space of S334ter-line-3 rats between 21 and 28 days of age. Responses to retinal stimulation were mapped in the SC of transplanted and sham control rats 78 to 163 days after surgery. The morphology of the retinas in all groups was examined. RESULTS: Photoreceptor cell loss in untreated rats matched the decline in visual activity in the SC. At 28 days, there was a scotoma in the area of the SC that represents the central retina and, by 63 days, it had enlarged to cover the entire retinal representation. Visual responses were evoked in 64% of rats with retinal transplants. These retinally driven responses were confined to a small, contiguous region of the SC that represents the sector of the retina where the transplant was placed. Visual responses were absent in the SC outside this area in transplant recipients and throughout the SC of untreated and sham control rats. CONCLUSIONS: Transplantation of fetal retinal sheets induced recovery of visual activity in the SC in this model of RP. The mechanisms underlying this functional recovery remain to be resolved, but these results suggest that transplantation should be further explored as a therapy for RP.     

Limitation of anatomical integration between subretinal transplants and the host retina

PURPOSE: In previous studies of subretinal transplantation in rabbits, the host photoreceptor layer seemed to prevent the bridging of neuronal fibers between the graft and the host retina. The current study was undertaken to determine whether the same phenomenon occurs in transplants to the subretinal space of the vascularized retina of rats. Bridging of fibers was examined in transplants to animals of different genetic backgrounds (normal versus dystrophic rats), of different ages, and after different survival times. METHODS: Sprague-Dawley (SD) rat retinal tissue from embryonic day (E)18 was subretinally grafted to adult (60-day-old) normal SD rats, to RCS rats (32 and 73 days old), and to adult (60-day-old) transgenic P23H rats. After various survival times (28-183 days), transplanted retinas were processed for routine histology and immunocytochemistry. Antibodies against calbindin, neuronal nitric oxide synthase (NOS), and protein kinase C (PKC) were used to identify specific retinal cell types and their processes. RESULTS: The shape and position of the immunoreactive cell bodies indicated that the expected neuronal populations were labeled within the grafts and in the host retina. Labeled neuronal processes were also observed. In each case, NOS-, calbindin-, and PKC-immunolabeled fibers formed bridges between the graft and the host tissues. However, regardless of the extent of host photoreceptor cell loss, the age of the recipient, or the genetic background, bridging fibers were observed only in areas where the host photoreceptor layer was discontinuous or completely missing. CONCLUSIONS: The present study demonstrates that the host photoreceptor layer plays a role in limiting graft-host anatomical integration.     

Differentiation of rat mesenchymal stem cells transplanted into the subretinal space of sodium iodate-injected rats

Purpose: The differentiation of rat bone marrow mesenchymal stem cells (MSCs) was investigated in a retinal pigment epithelium (RPE) damage model induced by the administration of sodium iodate. Methods: Cultured rat MSCs were transfected with enhanced green fluorescent protein and transplanted into the subretinal space of rats injected 4 days earlier with sodium iodate. Immunofluorescence analysis was performed 5 weeks later. Results: The transduction efficiency was 99.9%. Viable MSCs were detected 5 weeks after transplantation, mainly in the subretinal space. The cells expressed pan‐cytokeratin, glial fibrillary acidic protein and rhodopsin. Conclusions: Bone marrow MSCs transplanted into the subretinal space of sodium iodate‐injected rats have the ability to differentiate into RPE, photoreceptor and glial lineage cells.     

The fate of heterotopically grafted neural precursor cells in the normal and dystrophic adult mouse retina.

PURPOSE: To study the integration and differentiation of heterotopically transplanted neural precursor cells in the retina of adult mouse mutants displaying apoptotic degeneration of photoreceptor cells. METHODS: Neural precursor cells were isolated from the spinal cord of transgenic mouse embryos ubiquitously expressing enhanced green fluorescent protein. Cells were expanded in vitro and transplanted into the retina of adult wild-type and age-matched beta2/beta1 knock-in mice. Beta2/beta1 knock-in mutants display apoptotic death of photoreceptor cells and were generated by placing the cDNA of the beta1 subunit into the gene of the beta2 subunit of Na,K-ATPase. The integration and differentiation of grafted cells in recipient retinas was studied 1 or 6 months after transplantation. RESULTS: Mutant retinas contained more donor-derived cells than wild-type hosts. Moreover, in mutants, donor cells integrated into deeper retinal layers. In both genotypes, grafted cells differentiated into astrocytes and oligodendrocytes. Only a few ganglion cell axons were myelinated by donor-derived oligodendrocytes 1 month after transplantation, whereas extensive myelination of the nerve fiber layer was observed 6 months after transplantation. Unequivocal evidence for differentiation of grafted cells into neurons was not obtained. CONCLUSIONS: Heterotopically transplanted neural precursor cells are capable of integrating, surviving, and differentiating into neural cell types in normal and dystrophic retinas of adult mice. The particular environment of a pathologically altered retina facilitates integration of transplanted precursor cells. In principle, neural precursors may thus be useful to substitute for or replace dysfunctional or degenerated cell types. Results of the present study also indicate that replacement of retinal cell types is likely to require more appropriate donor cells, such as retinal precursor cells.     

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.     

骨髓间充质干细胞在视网膜色素变性大鼠视网膜下的分化

目的:研究骨髓间充质干细胞（MSC）在视网膜色素变性（RP）大鼠体内的分化。方法:Lewis大鼠腹腔注射30g/L NaIO₃ 100mg/kg,建立大鼠RP模型,将体外培养的MSC植入视网膜下腔,用免疫荧光标记的方法对MSC进行追踪,并观察术后第1,2,3,4,5wk MSC在该微环境中的分化。结果:术后第1wk即可见MSC位于视网膜色素上皮（RPE）层与光感受器细胞层,但全角蛋白（PCK）及rhodopsin标记阴性,第3wk开始可见MSC在体内表达PCK及rhodopsin。结论:MSC植入RP模型大鼠视网膜下腔后可存活,主要分布于RPE层和视锥、视杆细胞层,并表达RPE细胞和光感受器细胞的表面标志。     

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.     

Cyclic AMP prevents retraction of axon terminals in photoreceptors prepared for transplantation: an in vitro study

PURPOSE: Cell transplantation has emerged as a possible remedy for degeneration and injury in the central nervous system (CNS). In the retina, photoreceptor transplantation is a potential treatment for retinal degenerative disease. Graft survival has been well documented, but evidence of functional recovery is lacking. A major obstacle to recovery of vision is lack of synapse formation between grafted photoreceptors and host bipolar and horizontal cells. A prior study demonstrated that photoreceptors prepared for transplantation undergo rapid morphologic changes, including retraction of axon terminals toward their cell bodies, away from potential synaptic partners, a phenomenon that may interfere with graft-host synaptic interaction after transplantation. In this study, prevention of retraction of photoreceptor axon terminals was possible by pharmacological intervention. METHODS: Photoreceptor sheets, prepared by vibratome sectioning, and full-thickness retinas, harvested from adult porcine eyes, were maintained in culture and treated with either the cyclic adenosine monophosphate analogue 8-(4-chlorophenylthio)-cyclic 3',5'-adenosine monophosphate (CPT-cAMP), or forskolin, an adenylyl cyclase stimulant, for up to 48 hours. RESULTS: Both CPT-cAMP and forskolin treatments successfully blocked retraction of photoreceptor axon terminals. This effect was not due to cell toxicity and was reversed after removal of treatment, indicating its specificity. CONCLUSIONS: Pharmacological manipulation of photoreceptor axonal plasticity may improve graft-host synaptic interaction after subretinal photoreceptor cell transplantation.     

移植骨髓间充质干细胞治疗大鼠变性性视网膜病变

目的 观察视网膜下腔移植大鼠骨髓间充质干细胞(rMSCs)治疗碘酸钠诱发的变性性视网膜病变的效果.方法 Brown-Norway(BN)大鼠120只,分为碘酸钠注射模型组、rMSCs移植治疗模型组、正常对照组,每组各40只大鼠.模型组大鼠通过尾静脉注射碘酸钠建立变性性视网膜病变模型,正常对照组大鼠给予生理盐水注射.通过视网膜眼底照相、荧光素眼底血管造影、视网膜电图(ERG)和组织学方法鉴定视网膜色素上皮(RPE)和神经视网膜损伤,进一步使用原位凋亡检测(TUNEL)的方法对碘酸钠诱导视网膜变性的细胞病理学变化进行观察.原代分离rMSCs后进行流式细胞术鉴定,将CM-DiI荧光染料标记的rMSCs移植到受体动物的视网膜下腔,采用临床检查手段结合组织学检查方法对细胞疗法进行评估.在移植手术后14～60 d,检查rMSCs的存活,整合和分化情况.结果 在碘酸钠注射14 d内,模型组大鼠视网膜的功能逐渐衰竭,呈现时间依赖的关系.模型组大鼠RPE细胞破坏后,光感受器细胞外节出现断裂、缩短的变化直到核同缩.细胞核形态变化和TUNEL标记的结果表明光感受器细胞的死亡主要是凋亡.经过rMSCs移植.供体细胞能够存活并散在分布于视网膜下腔,分化为RPE细胞.ERG检查结果显示,60 d后模型组ERG b波改善率为27.80%,模型组ERG震荡电位(Ops)改善率为59.38%;表明rMSCs移植治疗模型组大鼠视网膜功能得到明显保护.结论 经过rMSCs移植,碘酸钠诱发的视网膜变性可以得到有效地治疗,移植后的rMSCs能够存活,分化为RPE细胞.     

Subretinal transplantation of bone marrow mesenchymal stem cells delays retinal degeneration in the RCS rat model of retinal degeneration

Because there is no effective treatment for this retinal degeneration, potential application of cell-based therapy has attracted considerable attention. Several investigations support that bone marrow mesenchymal stem cells (MSCs) can be used for a broad spectrum of indications. Bone marrow MSCs exert their therapeutic effect in part by secreting trophic factors to promote cell survival. The current study investigates whether bone marrow MSCs secrete factor(s) to promote photoreceptor cell survival and whether subretinal transplantation of bone marrow MSCs promotes photoreceptor survival in a retinal degeneration model using Royal College of Surgeons (RCS) rats. In vitro, using mouse retinal cell culture, it was demonstrated that the conditioned medium of the MSCs delays photoreceptor cell apoptosis, suggesting that the secreted factor(s) from the MSCs promote photoreceptor cell survival. In vivo, the MSCs were injected into the subretinal space of the RCS rats and histological analysis, real-time RT-PCR and electrophysiological analysis demonstrated that the subretinal transplantation of MSCs delays retinal degeneration and preserves retinal function in the RCS rats. These results suggest that MSC is a useful cell source for cell-replacement therapy for some forms of retinal degeneration.     

视网膜视细胞的成片移植

目的 探索用准分子激光切削技术制备视网膜单层细胞植片,经内入路视网膜下腔的单层视细胞成片移植。方法 用准分子激光对大鼠视网膜进行切削,制取单层视细胞植片,此后,按内入路手术方法进行了兔视网膜下腔的异种移植。结果 切削后所得视细胞植片由单层视细胞组成,结构完整,包括外丛状层、外核层和外节层;视细胞植片经明胶包埋后被准确植入宿主视网膜下腔中,移植术后第1,2天宿主观视网膜未能复位,呈脱离状态,移植物没能与视网膜色素上皮层相贴;移植后10天,宿主视网膜复位,视细胞移植片平铺于宿主视网膜下腔中,植片视细胞外节也宿主视网膜色素上皮层相贴;移植后10天,宿主视网膜复位,视细胞移植片平铺于宿主视网膜下腔中,植片视细胞外节与宿主视网膜色素上皮层相贴,未见明显免疫排异现象。结论 准分子激光制备单层视细胞植片方法简单、可行;初步观察到内入路单层视细胞成片移植后,视细胞植片能够在宿主视网膜下腔中以正常生理位置存活;视网膜下腔为理想的视网膜移植的受位。     

经巩膜外路至视网膜下腔移植视网膜细胞的实验研究

探讨视网膜光感受器细胞的移植方法及临床意义。方法将16只昆明鼠随机分为A组和B组,每组均8只鼠。于手术显微镜下,用特殊显微注射器穿过巩膜、脉络膜,在A组昆明鼠的视网膜下腔注入视网膜混合细胞,在B组昆明鼠的视网膜下腔注入纯光感受器细胞。于移植术后30、90及180 d摘除实验眼,于光镜下观察移植细胞在视网膜下腔生长的情况。结果大多数标本(13／15)HE染色显示视网膜细胞准确移植在受体眼的视网膜下腔,未见炎性细胞浸润和受体视网膜破坏;且移植到受体视网膜下腔的细胞在术后180 d仍存活。仅少数(2／15)标本可见受体视网膜结构破坏。移植的视网膜混合细胞均形成“玫瑰花”样结构,而移植的纯视网膜光感受器细胞则在视网膜下腔形成整齐的细胞层。结论经巩膜外路至视网膜下腔的显微注射法是较为理想的视网膜下腔注射给药和视网膜细胞移植方式。纯视网膜光感受器细胞移植后的生长状况和功能接近正常生理状态的视网膜组织结构,为临床治疗视网膜变性疾病提供了新途径。     

A new immunodeficient pigmented retinal degenerate rat strain to study transplantation of human cells without immunosuppression

Purpose

The goal of this study was to develop an immunodeficient rat model of retinal degeneration (RD nude rats) that will not reject transplanted human cells.

Methods

SD-Tg(S334ter)3Lav females homozygous for a mutated mouse rhodopsin transgene were mated with NTac:NIH-Whn (NIH nude) males homozygous for the Foxn1 ^rnu allele. Through selective breeding, a new stock, SD-Foxn1 Tg(S334ter)3Lav (RD nude) was generated such that all animals were homozygous for the Foxn1 ^rnu allele and either homo- or hemizygous for the S334ter transgene. PCR-based assays for both the Foxn1 ^rnu mutation and the S334ter transgene were developed for accurate genotyping. Immunodeficiency was tested by transplanting sheets of hESC-derived neural progenitor cells to the subretinal space of RD nude rats, and, as a control, NIH nude rats. Rats were killed between 8 and 184 days after surgery, and eye sections were analyzed for human, neuronal, and glial markers.

Results

After transplantation to RD nude and to NIH nude rats, hESC-derived neural progenitor cells differentiated to neuronal and glial cells, and migrated extensively from the transplant sheets throughout the host retina. Migration was more extensive in RD nude than in NIH nude rats. Already 8 days after transplantation, donor neuronal processes were found in the host inner plexiform layer. In addition, host glial cells extended processes into the transplants. The host retina showed the same photoreceptor degeneration pattern as in the immunocompetent SD-Tg(S334ter)3Lav rats. Recipients survived well after surgery.

Conclusions

This new rat model is useful for testing the effect of human cell transplantation on the restoration of vision without interference of immunosuppression.