Differences between the neurogenic and proliferative abilities of Müller glia with stem cell characteristics and the ciliary epithelium from the adult human eye

Much controversy has arisen on the nature and sources of stem cells in the adult human retina. Whilst ciliary epithelium has been thought to constitute a source of neural stem cells, a population of Müller glia in the neural retina has also been shown to exhibit neurogenic characteristics. This study aimed to compare the neurogenic and proliferative abilities between these two major cell populations. It also examined whether differences exist between the pigmented and non-pigmented ciliary epithelium (CE) from the adult human eye. On this basis, Müller glia with stem cell characteristics and pigmented and non-pigmented CE were isolated from human neural retina and ciliary epithelium respectively. Expression of glial, epithelial and neural progenitor markers was examined in these cells following culture under adherent and non-adherent conditions and treatments to induce neural differentiation. Unlike pigmented CE which did not proliferate, non-pigmented CE cells exhibited limited proliferation in vitro, unless epidermal growth factor (EGF) was present in the culture medium to prolong their survival. In contrast, Müller glial stem cells (MSC) cultured as adherent monolayers reached confluence within a few weeks and continued to proliferative indefinitely in the absence of EGF. Both MSC and non-pigmented CE expressed markers of neural progenitors, including SOX2, PAX6, CHX10 and NOTCH. Nestin, a neural stem cell marker, was only expressed by MSC. Non-pigmented CE displayed epithelial morphology, limited photoreceptor gene expression and stained strongly for pigmented epithelial markers upon culture with neural differentiation factors. In contrast, MSC adopted neural morphology and expressed markers of retinal ganglion cells and photoreceptors when cultured under similar conditions.This study provides the first demonstration that pigmented CE possess different proliferative abilities from non-pigmented CE. It also showed that although non-pigmented CE express genes of retinal progenitors, they do not differentiate into neurons in vitro, as that seen with Müller glia that proliferate indefinitely in vitro and that acquire markers of retinal neurons in culture under neural differentiation protocols. From these observations it is possible to suggest that Müller glia that express markers of neural progenitors and become spontaneously immortalized in vitro constitute a potential source of retinal neurons for transplantation studies and fulfil the characteristics of true stem cells due to their proliferative and neurogenic ability.     

Neural regeneration in the chick retina

In warm-blooded vertebrates, possibilities for retinal regeneration have recently become reality with the discovery of neural stem cells in the mature eye. A number of different cellular sources of neural stem cells have been identified. These sources include stem cells at the retinal margin, pigmented cells in the ciliary body and iris, non-pigmented cells in the ciliary body and Müller glia within the retina. This review focuses on recent reports of neural stem cells and regeneration in the postnatal chicken retina. In the chicken eye sources of neurogensis and regeneration include: (1) retinal stem cells at the peripheral edge of the retina; (2) Müller glia in central regions of the retina; (3) non-pigmented epithelial cells in the posterior portion of the ciliary body; and (4) possibly pigmented cells in the pars plana of the ciliary body. This review discusses the similarities between the retinal progenitor cells in the postnatal eye and those found in the embryo. In addition, I discuss combinations of growth factors, (insulin, IGF-I, EGF and FGF2) that are capable of stimulating the proliferation and production of neurons from neural progenitors, non-neural epithelial cells, and postmitotic support cells in the avian eye. In summary, the mechanisms that regulate the proliferation and differentiation of cells with neurogenic potential are beginning to be understood and the postnatal chicken eye has proven to be a useful model system to study retinal regeneration.     

The inhibitor of phagocytosis, O-phospho-l-serine, suppresses Müller glia proliferation and cone cell regeneration in the light-damaged zebrafish retina

The damaged zebrafish retina replaces lost neurons through a regenerative response that initiates with the asymmetric cell division of Müller glia to produce neuronal progenitor cells that proliferate and migrate to the damaged retinal layer, where they differentiate into the lost neuronal cell types. Because Müller glia are known to phagocytose apoptotic retinal cells during development, we tested if Müller glia engulfed apoptotic rod cell bodies in light-damaged retinas. After 24 h of constant intense light, damaged retinas revealed both a strong nuclear TUNEL signal in photoreceptors and a weak cytoplasmic TUNEL signal in Müller glia, although Müller glial apoptosis is not observed in the light-damaged retina. Light damage of a rod-specific transgenic reporter line, Tg(XlRho:EGFP)^fl1, resulted in some Müller glia containing both TUNEL signal and EGFP, which indicated that this subset of Müller glia engulfed apoptotic photoreceptor cell bodies.To determine if phagocytosis induced the Müller glial proliferative response in the light-damaged retina, we utilized O-phospho-l-serine (L-SOP), a molecule that mimics the phosphatidylserine head group and partially blocks microglial phagocytosis of apoptotic cells. Intravitreal injection of L-SOP immediately prior to beginning constant intense light treatment: i) did not significantly reduce light-induced photoreceptor cell death, ii) significantly reduced the number of PCNA-positive Müller glia, and iii) significantly reduced the number of cone photoreceptors in the regenerated retina relative to control retinas. Because L-SOP is also a specific group III metabotropic glutamate receptor (mGluR) agonist, we also tested if the more potent specific group III agonist, l-2-amino-4-phosphonobutyrate (L-AP4), the specific group III antagonist (RS)-α-Methylserine-O-phosphate (MSOP) or the specific group I antagonist, l-2-amino-3-phophonopropanoic acid (L-AP3) affected Müller glial proliferation. We found no changes with any of these factors compared to control retinas, revealing that metabotropic glutamate receptors were not necessary in the Müller glia proliferative response. Furthermore, ascl1a and stat3 expression were unaffected in either the L-SOP or MSOP-injected retinas relative to controls, suggesting L-SOP disrupts Müller glia proliferation subsequent to or in parallel with ascl1a and stat3 activation. This implies that at least one signaling mechanism, in addition to the process disrupted by L-SOP, is required to activate Müller glia proliferation in the light-damaged retina.     

The neurogenic competence of progenitors from the postnatal rat retina in vitro

The mammalian retina develops from stem or progenitor cells that are of neuroectodermal origin and derive from bilateral invaginations of the neuroepithelium, the optic vesicles. Shortly after birth, around 12 days postnatal in rats, the retina is fully developed in its cellular parts. Even though different cell types in the adult might be potential sources for retinal stem cells or progenitor cells, the retina is a non-neurogenic region and the diseased retina is devoid of any spontaneous regeneration. In an attempt to link late developmental processes to the adult situation, we analyzed the presence and the neurogenic potential of retinal progenitors during the postnatal period and compared it to adult ciliary body (CB) derived retinal progenitors and subventricular zone (SVZ) derived neural stem cells. Retinal progenitor properties were identified by the capacity to proliferate and by the expression of the progenitor markers Nestin, Flk-1, Chx10, Pax6 and the radial glia marker BLBP. The neurogenic potential was assayed by the expression of the neuronal markers doublecortin, betaIII Tubulin, Map2 and NSE, the glial makers A2B5, NG2, GalC and GFAP, and by incorporation of BrdU. The number of Flk-1 positive cells and concomitantly the number of newly born betaIII Tubulin-positive cells decreased within the first postnatal week in retinal progenitor cultures and no newly generated betaIII Tubulin, but GFAP positive cells were detected thereafter. In contrast to neural stem cells derived from the adult SVZ, postnatal and adult CB derived progenitors had a lower and a restricted proliferation potential and did not generate oligodendrocytes. The work demonstrates, however, that the existence of retinal progenitor cells is not restricted to embryonic development. In the sensory retina the differentiation potential of late retinal progenitors becomes restricted to the glial lineage, whereas neurogenic progenitor cells are still present in the CB. In addition, major differences in growth and differentiation potential of adult neural stem cells and postnatal and adult retinal progenitors are presented.     

Changes in Musashi-1 subcellular localization correlate with cell cycle exit during postnatal retinal development

RNA-binding proteins, and in particular, the Musashi genes, function as essential regulators of progenitor functioning in both the developing and adult organism. In this report, we characterize the differential subcellular distribution of Musashi-1 in cells engaged in either proliferating or differentiating contexts in the developing mouse retina, and in cultured Müller glia. During retinal cell differentiation, Musashi-1 immunoreactivity shifts from exclusively cytoplasmic in retinal progenitor cells, to predominantly nuclear localization in differentiating neurons. This nuclear shift is transient, with localization in the adult retina becoming predominantly perinuclear and cytoplasmic in Müller glia and photoreceptors. A correlation between cell cycle progression and subcellular distribution of Musashi-1 is observed in passageable, adult Müller glial cells in vitro. Furthermore, treatment of Müller cultures with neuron-promoting differentiation media induces asymmetric cytoplasmic Musashi-1 immunoreactivity in dividing daughter cells. The observed shifts in subcellular Musashi-1 localization are consistent with contrasting roles for Musashi-1 during cell proliferation and differentiation. These data provide evidence that nuclear, and cytoplasmic sequestering of Musashi-1 in retinal cells is context-specific, and may contribute to downstream functioning of Musashi-1.     

CNTF induces photoreceptor neuroprotection and Müller glial cell proliferation through two different signaling pathways in the adult zebrafish retina

Ciliary neurotrophic factor (CNTF) acts in several processes in the vertebrate retina, including neuroprotection of photoreceptors in the stressed adult retina and regulation of neuronal progenitor cell proliferation during retinal development. However, the signaling pathway it utilizes (Jak/Stat, MAPK, or Akt) in these processes is ambiguous. Because dark-adapted albino zebrafish exhibit light-induced rod and cone cell death and subsequently regenerate the lost photoreceptor cells, zebrafish should be a useful model to study the role of CNTF in both neuroprotection and neuronal progenitor cell proliferation. We therefore investigated the potential roles of CNTF in both the undamaged and light-damaged adult zebrafish retinas. Intraocular injection of CNTF suppressed light-induced photoreceptor cell death, which then failed to exhibit the regeneration response that is marked by proliferating Müller glia and neuronal progenitor cells. Inhibiting the MAPK signaling pathway, but neither the Stat3 nor Akt pathways, significantly reduced the CNTF-mediated neuroprotection of light-induced photoreceptor cell death. Intraocular injection of CNTF into non-light-treated (undamaged) eyes mimicked constant intense light treatment by increasing Stat3 expression in Müller glia followed by increasing the number of proliferating Müller glia and neuronal progenitors. Knockdown of Stat3 expression in the CNTF-injected non-light-treated retinas significantly reduced the number of proliferating Müller glia, while coinjection of CNTF with either MAPK or Akt inhibitors did not inhibit the CNTF-induced Müller glia proliferation. Thus, CNTF utilizes a MAPK-dependant signaling pathway in neuroprotection of light-induced photoreceptor cell death and a Stat3-dependant signaling pathway to stimulate Müller glia proliferation.     

Müller cells express the neuronal progenitor cell marker nestin in both differentiated and undifferentiated human foetal retina

Tritiated thymidine studies suggest that Müller cells are the last cells born in the retina, although several authors describe Müller cells throughout the retina from very early ages. In this study immunohistochemistry was used to identify progenitor and Müller cells in human foetal retina. Antibodies to nestin (an intermediate filament protein expressed by neural progenitor cells), vimentin, cellular retinaldehyde binding protein (CRALBP) and glutamate and aspartate transporter (GLAST), which are each expressed by Müller cells, were used in combination with anti-Ki67 to identify proliferating cells. By definition, Ki67-positive proliferating cells were present in undifferentiated retina, but not in differentiated retina. Nestin-immunoreactive (IR) cells colocalized with vimentin throughout the retina. CRALBP-IR was detected in differentiated retina and in some proliferating cells. GLAST-IR cells were present only within the differentiated region. Nestin, vimentin and CRALBP each colocalized with mitotic Ki67-IR cells, suggesting that in foetal retina Müller cells and retinal progenitor cells are overlapping populations and that Müller cells are end-stage progenitor cells.     

A role for DNA methylation in regulation of EphA5 receptor expression in the mouse retina

Understanding the mechanisms regulating expression of retinal ganglion cell (RGC) specific and axon-guidance genes during development and in retinal stem cells will be critical for successful optic nerve regeneration. Müller glia have some characteristics of retinal stem cells but in mammals have demonstrated limited potential to differentiate into RGCs. Chromatin remodeling through histone deacetylation and DNA methylation are a potential mechanism for silencing genes necessary for neuronal differentiation of glial cells. We investigated DNA methylation as a mechanism for regulating expression of mouse EphA5, one member of a large family of ephrin receptor genes that regulate patterning of the topographic connections of RGCs during visual system development. We analyzed spatial and age-related patterns of EphA5 promoter methylation by bisulfite sequencing and mRNA expression by quantitative RT-PCR in the mouse retina. The CpG island in the EphA5 promoter was hypomethylated in the retina and showed no change in overall methylation with age, despite a decline in EphA5 mRNA expression levels in the adult retina. In the nasal retina of post-natal day 0 mice, there was a modest, but statistically significant increase in methylation. Increased methylation corresponded with lower levels of receptor mRNA expression in the nasal retina. We cloned the EphA5 promoter and found that site-specific differences in methylation could preferentially activate or repress promoter activity in transient transfections of rat retinal progenitor cells (R28) using luciferase assays. In sphere cultures generated by EGF/FGF2 stimulation of conditionally immortalized mouse Müller glia (ImM10), EphA5 promoter was hypermethylated and EphA5 mRNA was not detected. Demethylation using 5-azadeoxycytidine (AzadC) resulted in a significant decrease of methylation of the EphA5 promoter and re-expression of the EphA5 mRNA. The inverse relationship between EphA5 promoter methylation and mRNA expression is consistent with a role for DNA methylation in modulating the spatial patterns of EphA5 gene expression in the retina and in silencing EphA5 expression in ImM10 cells. The robust up-regulation of EphA5 in ImM10 cells following demethylation suggests that modulation of chromatin structure may be a useful approach for promoting expression of silenced developmental genes and increasing the neurogenic potential of Müller glia.     

四种中间丝蛋白在大鼠视网膜发育过程中表达的时间顺序和结构特征

目的研究视网膜内神经干细胞的标志物及其表达的时间和空间分布特征。方法利用免疫组化染色,检测四种中间丝蛋白(Nestin、Vimentin、GFAP及NF)在新生至12月龄大鼠视网膜内表达的时间顺序、空间分布和强度变化。结果大鼠视网膜结构的分化要到出生后2周才能完成。在2周内Nestin的表达可见于视网膜的神经细胞、胶质细胞、血管内皮细胞内。2周后表达水平明显降低。4周时完全转阴。但在睫状体的少数突起内,Nestin持续高水平表达,成年时,阳性细胞以色素上皮细胞为主。此外,在成年大鼠的视神经内也可见散在的Nestin阳性细胞。Vimentin在神经胶质细胞和睫状体的无色素上皮细胞中一直呈强阳性。除了文献报道的水平细胞外,我们还发现Vimentin在4周内的MUller细胞和无长突细胞内表达。GFAP和NF仅在发育成熟的神经胶质细胞和神经节细胞的轴突内表达。结论Nestin可作为视网膜神经干细胞的标志,成年时视网膜的神经干细胞分布在少数睫状突及视神经内。（中华眼科杂,2004,40：765—769)     

Müller glia: Stem cells for generation and regeneration of retinal neurons in teleost fish

Adult zebrafish generate new neurons in the brain and retina throughout life. Growth-related neurogenesis allows a vigorous regenerative response to damage, and fish can regenerate retinal neurons, including photoreceptors, and restore functional vision following photic, chemical, or mechanical destruction of the retina. Müller glial cells in fish function as radial-glial-like neural stem cells. During adult growth, Müller glial nuclei undergo sporadic, asymmetric, self-renewing mitotic divisions in the inner nuclear layer to generate a rod progenitor that migrates along the radial fiber of the Müller glia into the outer nuclear layer, proliferates, and differentiates exclusively into rod photoreceptors. When retinal neurons are destroyed, Müller glia in the immediate vicinity of the damage partially and transiently dedifferentiate, re-express retinal progenitor and stem cell markers, re-enter the cell cycle, undergo interkinetic nuclear migration (characteristic of neuroepithelial cells), and divide once in an asymmetric, self-renewing division to generate a retinal progenitor. This daughter cell proliferates rapidly to form a compact neurogenic cluster surrounding the Müller glia; these multipotent retinal progenitors then migrate along the radial fiber to the appropriate lamina to replace missing retinal neurons. Some aspects of the injury-response in fish Müller glia resemble gliosis as observed in mammals, and mammalian Müller glia exhibit some neurogenic properties, indicative of a latent ability to regenerate retinal neurons. Understanding the specific properties of fish Müller glia that facilitate their robust capacity to generate retinal neurons will inform and inspire new clinical approaches for treating blindness and visual loss with regenerative medicine.     

Prospects for the application of Müller glia and their derivatives in retinal regenerative therapies

Neural cell death is the main feature of all retinal degenerative disorders that lead to blindness. Despite therapeutic advances, progression of retinal disease cannot always be prevented, and once neuronal cell damage occurs, visual loss cannot be reversed. Recent research in the stem cell field, and the identification of Müller glia with stem cell characteristics in the human eye, have provided hope for the use of these cells in retinal therapies to restore vision. Müller glial cells, which are the major structural cells of the retina, play a very important role in retinal homeostasis during health and disease. They are responsible for the spontaneous retinal regeneration observed in zebrafish and lower vertebrates during early postnatal life, and despite the presence of Müller glia with stem cell characteristics in the adult mammalian retina, there is no evidence that they promote regeneration in humans. Like many other stem cells and neurons derived from pluripotent stem cells, Müller glia with stem cell potential do not differentiate into retinal neurons or integrate into the retina when transplanted into the vitreous of experimental animals with retinal degeneration. However, despite their lack of integration, grafted Müller glia have been shown to induce partial restoration of visual function in spontaneous or induced experimental models of photoreceptor or retinal ganglion cell damage. This improvement in visual function observed after Müller cell transplantation has been ascribed to the release of neuroprotective factors that promote the repair and survival of damaged neurons. Due to the development and availability of pluripotent stem cell lines for therapeutic uses, derivation of Müller cells from retinal organoids formed by iPSC and ESC has provided more realistic prospects for the application of these cells to retinal therapies. Several opportunities for research in the regenerative field have also been unlocked in recent years due to a better understanding of the genomic and proteomic profiles of the developing and regenerating retina in zebrafish, providing the basis for further studies of the human retina. In addition, the increased interest on the nature and function of cellular organelle release and the characterization of molecular components of exosomes released by Müller glia, may help us to design new approaches that could be applied to the development of more effective treatments for retinal degenerative diseases.     

Expression of Cre recombinase in retinal Müller cells

Purpose

In an effort to generate inducible RPE-specific Cre mice using a 3.0-kb human vitelliform macular dystrophy-2 (VMD2) promoter, we identified a mouse line with unanticipated Cre activity in the neural retina, including Müller glial cells. Müller cells play important roles in the function and maintenance of the retina, and this mouse line would be potentially useful for conditional gene targeting in Müller glia. We therefore characterized the timing, inducibility, and cell specificity of Cre expression, as well as Müller cell-specific efficiency of Cre-mediated recombination in this mouse line.

Methods

Transgenic mice carrying cassettes of human P_VMD2-rtTA and TRE-cre were generated. Cre expression was characterized using a Cre-activatable lacZ reporter mouse line (R26R) and a floxed interleukin six signal transducing receptor (gp130) mouse line.

Results

β-Galactosidase (β-gal) assay and immunohistochemical analysis of VMD2-cre/R26R double transgenic mice indicated that Cre activity was detected in cells located in the inner nuclear layer, with prominent expression of β-gal in Müller cells. Cre activity was also detected in photoreceptors in the outer nuclear layer. PCR analysis demonstrated that Cre-mediated recombination initiated by embryonic day 15. Immunohistochemical analysis indicated that Cre-mediated deletion of floxed gp130 gene occurred in 52% of the retinal Müller cells. Retinal function and morphology were normal in 10-month-old VMD2-cre mice.

Conclusion

We generated a transgenic cre mouse that is useful to study gene activation and inactivation in retinal Müller cells.     

Purinergic signaling involved in Müller cell function in the mammalian retina

Purines (in particular, ATP and adenosine) act as neuro- and gliotransmitters in the sensory retina where they are involved in bidirectional neuron-glia signaling. This review summarizes the present knowledge about the expression and functional importance of P1 (adenosine) and P2 (nucleotide) receptors in Müller glial cells of the mammalian retina. Mammalian Müller cells express various subtypes of adenosine receptors and metabotropic P2Y receptors. Human Müller cells also express ionotropic P2X₇ receptors. Müller cells release ATP upon activation of metabotropic glutamate receptors and/or osmotic membrane stretching. The osmotic mechanism is abrogated under conditions associated with ischemia-hypoxia and inflammation, resulting in swelling of the Müller cells when the extracellular milieu is hypoosmotic. However, exogenous glutamate, which induces the release of ATP and adenosine, and thus activates P2Y₁ and A₁ adenosine receptors, respectively, prevents such osmotic swelling under pathological conditions, suggesting unimpaired receptor-induced release of ATP. In addition to the inhibition of swelling, which is implicated in regulating the volume of the extracellular space, purinergic signaling is involved in mediating neurovascular coupling. Furthermore, purinergic signals stimulate the proliferation of retinal precursor cells and Müller cells. In normal retinal information processing, Müller cells regulate the synaptic activity by the release of ATP and adenosine. In retinopathies, abrogation of the osmotic release of ATP, and the upregulation of ecto-apyrase (NTPDase1), may have neuroprotective effects by preventing the overactivation of neuronal P2X receptors that are implicated in apoptotic cell death. Pharmacological modulation of purinergic receptors of Müller cells may have clinical importance, e.g., for the clearance of retinal edema and for the inhibition of dysregulated cell proliferation in proliferative retinopathies.     

Expression and localisation of BDNF, NT4 and TrkB in proliferative vitreoretinopathy

Exogenous brain derived neurotrophic factor (BDNF) is known to rescue ganglion cell death after optic nerve injury. Its mechanism of action is believed to be indirect via glial cells in the retina. In this study we investigated the changes in expression and localisation of BDNF, neurotrophin-4 (NT4) and their common receptor (TrkB) in retinectomy sections of patients with proliferative vitreoretinopathy (PVR). Nine full-thickness retinectomy specimens obtained at retinal reattachment surgery for PVR were fixed in 4% paraformaldehyde immediately after excision and compared to similarly processed normal donor retinas (4 eyes). Agarose-embedded sections (100 microm thick) were double labelled for immunohistochemistry by confocal microscopy, with antibodies against BDNF, NT4, TrkB, rod opsin, glial fibrillary acidic protein (GFAP), cellular retinaldehyde binding protein (CRALBP) and Brn3. This study demonstrates expression of NT4 by ganglion cells and shows expression of BDNF and NT4 in the outer photoreceptor segments is downregulated during PVR, whilst NT4 is markedly upregulated throughout the retina during this condition. The findings here suggest that NT4 may play a neural protective role during the development of PVR. It also shows that upregulation of NT4 in PVR is localised to Müller glial cells, indicating either over-expression of this factor by Müller cells or that Müller cells internalise NT4 for trafficking across the retina. TrkB expression was not observed in PVR retina. The observations that Müller glia demonstrate upregulation of NT4 suggests that retinal injury may lead to activation of this neurotrophin by Müller cells as part of their neuroprotective functions.     

视网膜内源性干细胞研究进展

视网膜的神经细胞一旦损伤,便无法再生修复,利用干细胞疗法使神经细胞得到再生修复已成为当今研究的热点.视网膜中存在具有自我修复能力的内源性干细胞,激活视网膜中的内源性干细胞,利用其对损伤的视网膜神经元进行修复,具有重要的研究价值和应用前景,近年来受到世界神经生物学、眼科学等领域研究者的重视.鱼类、两栖类动物的视网膜具有较强的再生修复能力,而鸟类和哺乳类动物的视网膜的再生能力有限,且各类动物的视网膜再生存在各自的特征.睫状体边缘带、视网膜色素上皮细胞、Müller细胞等细胞都是视网膜再生可能的细胞来源,如鱼类、鸟类和哺乳类动物新生成的视网膜细胞来自于视网膜Müller细胞,而两栖类动物再生的视网膜则来源于视网膜色素上皮细胞.各种视网膜内源性干细胞需要被活化后才能发挥干细胞特性对损伤的视网膜神经细胞进行修复,激活这些细胞的方法有多种,如利用兴奋性氨基酸、生长因子、转录因子、细胞内信号等.本文就不同物种,包括鱼类、两栖类、鸟类和哺乳类动物在内的视网膜的再生能力、睫状体边缘带、视网膜色素上皮细胞、Müller细胞等不同的视网膜内源性神经干细胞的来源及各种能够活化视网膜内源性干细胞进行增生和分化的因素的相关研究进展进行综述.     

体外诱导大鼠视网膜Müller细胞向神经节细胞定向分化的研究

目的 研究鼠视网膜Müller细胞经体外条件培养基诱导后去分化为神经干细胞及进一步定向分化成神经节样细胞的特性.方法 实验研究.体外培养出生后7-10 d的Sprague Dawley大鼠视网膜Müller细胞,应用逆转录聚合酶链反应(RT-PCR)法及免疫荧光染色法鉴定Müller细胞纯度.取培养的第3或4代Müller细胞,用含有N2、碱性成纤维细胞生长因子和表皮生长因子的Delbeccon's modified Eagle's medium(DMEM)-F12干细胞条件培养基培养3～5 d后,再用含5%胎牛血清、脑源性神经营养因子和视黄酸的DMEM培养基诱导分化7～10 d,采用免疫荧光染色法分别鉴定去分化及再诱导分化后的细胞.结果 RT-PCR及免疫荧光染色结果显示,分离培养的视网膜Müller细胞纯度可达(95.17±2.68)%以上.干细胞条件培养基培养3～5 d后,大部分Müller细胞汇集形成细胞球,经免疫荧光染色法鉴定,显示细胞球内(95.26 ±1.35)%以上的细胞Nestin表达阳性,(90.33±4.12)%以上细胞BrdU表达阳性.将这些细胞球进一步诱导分化后,可见细胞球内细胞可向外扩展、铺开,分化出形态各异的细胞,其中约(21.14±1.49)%细胞表达神经节细胞特异性标记物Thy1.1阳性.结论 成年啮齿动物视网膜Müller细胞经体外条件培养基诱导后,可以产生具有增殖能力和分化潜能的神经干细胞,并可进一步定向分化为神经节样细胞,这为干细胞研究和视神经再生治疗等提供了新的方法和手段.     

Cones regenerate from retinal stem cells sequestered in the inner nuclear layer of adult goldfish retina

PURPOSE: To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated cones after laser ablation of photoreceptors in adult goldfish retina. METHODS: Using a technique developed previously in this laboratory, photoreceptors in the retina of adult goldfish were ablated with an argon laser. The mitotic marker, bromodeoxyuridine, was used to label proliferating and regenerated cells, which were identified with cell-specific markers. RESULTS: Cells proliferating locally within lesion included microglia, Müller glia, and retinal progenitors in the inner nuclear layer (INL). The nuclei of both Müller glia and associated retinal progenitors migrated from the inner to the outer nuclear layer. The proliferating retinal progenitors, which express Notch-3 and N-cadherin, regenerated cone photoreceptors and then rod photoreceptors. CONCLUSIONS: Previous work has demonstrated that photoreceptors in the goldfish retina regenerate selectively after laser ablation, but the source of regenerated cones has not been identified. The results reported here provide support for the existence of retinal stem cells within the adult fish retina that are capable of regenerating cone photoreceptors. The data also support the involvement of Müller glia in the production of regenerated cones.     

Nestin positive cells in adult human retina and in epiretinal membranes

BACKGROUND/AIM: Nestin is an intermediate filament marker for neural progenitor cells. The authors aimed to identify nestin positive cells in adult human retina and within surgically removed epiretinal membranes. METHODS: Adult human retina and epiretinal membranes were studied. Tissue was fixed and processed for semithin sections or whole mount preparations for immunohistochemical detection of nestin and glial fibrillary acidic protein (GFAP) expression. RESULTS: Nestin positive cells are most prominent at the ora serrata, possess fibrillary processes, small amounts of perinuclear cytoplasm, and are arranged radially within or superficially on the retina. In the posterior retina, speckled cytoplasmic nestin staining is seen around the nuclei of neurons. In the peripapillary retina most of the cells in the retinal ganglion cell layer are nestin positive. These cells appear to represent nestin positive neurons. Speckled cells are also seen in the myelinated portion of the optic nerve. In epiretinal membranes patches of elongated nestin positive cells were found. These cells were also positive for GFAP. CONCLUSIONS: Some neurons and glia in the adult human retina are nestin positive. Their pattern in anterior retina suggests an analogy with the ciliary marginal zone found in many other species. The role of these cells in pathological responses to retinal disease is suggested by the presence of large numbers of ectopic nestin positive cells in epiretinal membranes. The authors hypothesise that nestin positive cells represent a population of progenitor cells from normal adult human retina that differentiate to make up retinal scar tissue.