Cell based interventions may provide treatment avenues for these retinal degenerative conditions. Ongoing research findings in regenerative medicine have raised hopes of people suffering from brain diseases such as Parkinson's disease, stroke, Alzheimer's disease, ALS and spinal cord injury. Stem cells whether hematopoietic,2, 12, 13 mesenchymal,18-21 embryonic,22, 23 neural stem cells,15, 24-28 retinal progenitors,14, 16, 29-31 have been studied for their differentiation potential into neuronal,32 astrocytic,12, 24 as well as various retinal cell lineages.14, 15, 20, 23, 27, 33 Stem cell tranplantation therapy is attracting attention for chronic diseases of ageing and may provide a solution to such disorders.33 It can provide a potential strategy for replacing damaged neurons and restoring their functional activity. Recent studies demonstrate that SCT to the site of neural degeneration can facilitate differentiation into neurons, resulting in motor and cognitive improvements in animal models.34 The use of umbilical cord blood (UCB) derived stem cells have been attempted with various disease models such as stroke,35 Alzheimer's,36 spinal cord injury,37 cardiomyopathy38 and ischemic limb disorder.39
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A zone of proliferating retinal stem cells exists at the far peripheral edge of the retina of fish, frogs and birds. This zone of stem cells has been termed the ciliary or circumferential marginal zone (CMZ). In lower vertebrates, larval frogs, embryonic chicks, urodeles, ambhibians and fish, it has been identified that, ciliary marginal zone is one of the prominent source of resident stem cell population involved in regeneration of the injured or degenerated retina.40-42 Mammals are known to lack CMZ, which is a potential stem source in lower vertebrates. In the process of evolution, mammals appear to have lost ciliary marginal zone even though a quiescent population of stem cells do exist in pigmented ciliary epithelium. Factors that are responsible for quiescence, localization of CE stem cells and function of quiescent stem cells in normal eye need to be observed.43 Since the identification of CE stem cells, several groups have studied the response of ciliary epithelial stem cells present at the periphery of the retina in mammalian retinal injury models. However, only a few groups have studied the response of ciliary epithelial stem cells upon activation of certain signaling pathways in retinal injury models. Though significant work has been done in this area, many aspects in understanding the nature of these stem cells can be derived from transplantation experiments.
Laser induced mouse model has been frequently used for understanding the pathophysiology of AMD. This model provides reproducible platform for pre-clinical screening of biotherapeutics aided by laser photocoagulator. This hypothesis proposes to establish the model and compare the efficacy of lin-ve UCB derived stem cells and the neurospheres derived from abortus. The proposed study has been designed to investigate the comparative recruitment of fetal ciliary epithelium derived neurospheres to that of lin-ve UCB derived stem cells at the site of laser induced injury when transplanted through subretinal routes. Both lin-ve UCB derived stem cells and Ciliary epithelium derived neurospheres would be transplanted in mice with retinal laser injury through subretinal route and their homing and differentiation capacity would be investigated at varying time points.
The current literature in the area of regenerative medicine suffers from a lack of comparative data between types of stem cells, dose response and routes of delivery. Lately, there has also been over emphasis on the studies examining autologous sources of stem cells including BMSCs and UCB derived cells. The proposed study has been designed to address this deficit by investigating the comparative recruitment of human fetal ciliary epithelium derived neurospheres to that of Bone marrow derived lin-ve stem cells at the site of laser induced injury when transplanted through subretinal route. Both UCB derived Lin-ve stem cells and CE derived neurospheres have not been compared for their efficacy thus undermining the pace and quality of clinical translation.
This study serves to compare the efficacy of these two sources of stem cells by using standard molecular tools and electrophysiological end points. This study will also shed light on whether stem cells from local origin provide better substrate for cellular therapy in retinal degeneration or lin-ve UCB cells which are of haematopoeitic origin.
The vertebrate retina provides an ideal model for investigations of central nervous system (CNS) development and plasticity. It is organized into distinct, well-defined layers and, because of its localization it is readily accessible for experimental manipulation and analysis. Besides, the cellular organization and gross morphogenesis of the eye and retina are similar across vertebrate species. Although synaptic organization of retina is similar as compared to that of the other central neural structures, it is however relatively simpler than other brain regions. Retina has therefore been a platform of choice for conducting CNS transplantation experiments by neural stem cell biologists to study neural development and regeneration. The cytoarchitectural organization of the mature retina results from a complex series of developmental processes involving both intrinsic and extrinsic cues. A large body of literature exists in which the processes of neurogenesis and differentiation within the retina have been described in considerable detail.44,45 In addition, wide range of species have been used as model systems for studying retinal and visual system development, each system being of comparative interest because of its own unique attributes. Retina contains five major classes of neurons that are linked in an intricate pattern of connections but with an orderly layered anatomical arrangement.
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Mammalian retina is composed of seven distinct layers consisting of different neurons and glia that include, ganglion cells in ganglion layer (GCL - innermost layer), bipolar cells, amacrine cells and muller glia in inner nuclear layer (INL), which act as inter neurons, rod and cone photoreceptors in the photoreceptor layer (PRL) also considered as outer nuclear layer (ONL) and retinal pigment epithelial layer. The axons of ganglion cells traverse through optic nerve and connect to visual cortex of the brain. The rods function in conditions of low illumination whereas cones are responsible for color vision and various visual tasks that require high resolution, such as reading. Most of the mammalian species have rods with a small sized cell body and long and slender inner and outer segment architecture. Their outer segments are particularly thin to penetrate into the processes of pigment epithelial cells. The rods are located away from the center of the eye and are mostly concentrated in the retinal periphery and therefore responsible for peripheral vision. Cones are concentrated at a specialized central temporal area of the retina in many mammalian species, known as macula. Horizontal cells are the second order neurons interconnecting photoreceptor terminals laterally across the plane of the retina. Their role in vision is to modulate the vertical pathways from photoreceptors to bipolar cells in both a feedback and feed forward manner. Horizontal cells have varying morphologies adapted to deal best with the particular associated photoreceptors. Bipolar cells play their role in vision by establishing a communication link with photoreceptors in the outer plexiform layer and ganglion cells in the inner plexiform layer. Neurophysiologically these cells are divided into two types:that depolarize in response to central or direct illumination and hyperpolarizing center bipolar cells that hyperpolarize in response to center illumination. Both of these types form synaptic connections with ganglion cells.these are the final output neurons of the retina carrying the visual information that has been processed by the neurons of the retina to higher centers of visual processing in brain. Amacrine cells share functional similarity to the interneurons in the CNS. They control the excitatory pathway of the retina's 'principal neurons' bipolar and ganglion cells by forming synaptic contacts with these cells. They provide a pathway for both narrow and wide field lateral interactions within the IPL. The cell bodies of amacrine cells lie in the proximal part of the INL. the RPE performs some of the most important functions for the survival and maintenance of neural retina. It performs phagocytosis of shed fragments of photoreceptor outer segments 47-49 and secretes growth factors like VEGF and PEDF that help in the survival and growth of retinal cells. It provides a transport system for movement of water molecules, ions (K+, Cl-) and removal of metabolic end products between sub-retinal space and the blood.50-54 RPE also provides nutritional support to retina by carrying out transport of glucose, fatty acids, amino acids, ascorbic acid and retinol form blood to photoreceptors. The RPE prevents aberrant vision by absorbing light energy focused by lens on the retina.46, 55 There are a number of diseases in which photoreceptors and other cellular components of neural retina get affected at one or the other stage of the disease. The most common among all the diseases responsible for vision loss are inherited retinal degenerations, age-related macular degeneration and glaucoma. The final outcome of these diseases is usually complete or partial loss of vision depending upon the type and stage of disease.
Retinal degeneration describes damage or atrophy of the retina that may arise due to a vascular disease, exudative or inflammatory damage, ageing, mechanical forces, trauma or toxicity. Retinitis pigmentosa is the most common type of retinal degeneration and it describes a group of hereditary disorders that affect retina and characterized by progressive dysfunction of photoreceptors. Unlike refractive anomalies such as myopia or astigmatism, retinal degenerations cannot be corrected with lenses, and in most cases they cannot be treated.
Macular degeneration is the major cause of vision loss in people over the age of 50 in developing countries. It belongs to a very poorly understood group of diseases that cause sight-sensing cells i.e. cones, in the macular zone of the retina to malfunction. The result is debilitating loss of vital central or detail vision. Macular degeneration is divided into two broad categories: early onset and age-related. AMD is further divided into wet (neovascular) and dry types (geographic atrophy). Dry ARMD contributes 80% of macular degeneration and is characterized by a number of histopathological changes such as drusen formation and basal laminar deposits, RPE and photoreceptor degeneration and inability to function properly and support other retinal neurons. Wet form of ARMD is more severe and is characterized by leaky blood vessel formation that occludes vision. Over the time the degenerated RPE causes the photoreceptors to function improperly, resulting in loss of vision. Glaucoma is the second major leading cause of blindness. In glaucomatous patients, the blood supply to the optic nerve head is altered, inducing ischemia which in turn affects normal ganglion cell axon function. Axonal injury results in retrograde neuronal degradation due to insufficient neurotrophic factors. A characteristic feature of ganglion cell death in glaucoma is its slow and variable nature which makes it more likely that cell based interventions could be useful in this disease. Retinitis Pigmentosa (RP) addresses a vast group of RDs that arise as a result of mutations in various visual machinery components. Such degenerations are characterized by progressive night blindness, visual field loss, optic nerve atrophy, arteriolar attenuation, altered vascular permeability and central loss of vision that often ends up in complete blindness. There have been a number of genes identified that are defective in individuals suffering from these diseases. The worst affected retinal components in these diseases are the enzymatic and structural components of the phototransduction network. These include rhodopsin,56,57 Peripherin,58 and RPE65.59 Exposure to certain high energy lasers can also result in injury or burn in eyes and skin of the person exposed to laser. The laser light in most of commercial lasers such as laser pointers is not powerful enough to cause damage to skin. However, lens in our eye focuses the light entering into it onto the retina making retina very vulnerable to almost any type of laser. The energy focused onto the retina can be many times greater than that falling on the cornea. Therefore, depending upon the type of laser, energy output of laser and distance from the laser source, there can be various types of injuries ranging from tiny lesions to severe burns. Laser weapons have been developed to damage battlefield electro-optical sensors and visually incapacitate soldiers.60, 61 The increasingly widespread use of lasers in clinics and industry has demanded an effective and successful treatment therapy for laser eye injuries. Laser photocoagulation is routinely used in various treatment methods by ophthalmologists and cosmetic surgeons. An accidental exposure of eyes to laser may result in either immediate or progressive vision loss. One of the existing treatment strategies for laser injured retina is the use of corticosteroids to reduce cellular inflammation.Stem cells are pluripotent and have ability to self-renew and give rise to multiple mature cell types. The two important characteristics that distinguish them from other types of cells are: i) they are unspecialized cells that renew themselves for long periods through cell division, ii) under certain physiologic or experimental conditions, they can be induced to become cells with special functions such as those of the heart muscle or the insulin-producing cells of the pancreas. A major breakthrough in the field of neurobiology was the demonstration that immature progenitor cells with multiphenotypic potential can be isolated from the CNS of both the developing and adult rodents and propagated for long periods in culture.65
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Stem cells when transplanted into eyes with retinal damage, undergo migration and integrate into various retinal layers and express retinal neural markers.64 Sclera and choroid cells isolated form adult human eyes have also been reported to possess the potential to differentiate towards the neural lineage.62 The differentiation of stem cells into viable photoreceptors and their successful transplantation has been shown in dystrophic mice.70 Retinal neurons cultured from hESCs and cell lines have shown long term survival and integration post subretinal transplantation.66,67The cell replacement depends on the type of cells that are damaged originally and on the presence of growth factors such as FGF-2, VEGF, BDNF and insulin. The stem cells in the recent past have shown advancements not only for transplantations but also as vectors for the transportation of growth factors, neurotropic factors and administration of gene therapy vectors as well.68,69 Advancements in studies in the field of regenerative medicine has paved a path for the evolution of state of the art techniques in minimizing the graft rejection. Subretinal injections have emerged as the most successful route for cell migration and incorporation into the various layers of the retina. Subretinal injections have been advanced to the extent of transplantations of ultrathin substrates of cells as well as tissue ensheathings.71,72
The dawn of induced pluripotent stem cells have given further hopes for clinical translation. The retinal neurons, RPE cells and photoreceptors can be generated from these cells and can be used in human translation.73 A recent clinical trial with a subretinal prosthesis exhibited visual restoration, inferring that even a severe degenerations in the retina may be repaired after cell replacement through potential plasticity of the visual system. Successful differentiation of neural retina from induced pluripotent stem cells (iPSCs) and the recent generation of an optic cup from human Embryonic stem cells in-vitro has increased the possibilities of generating clinically suitable source of cells for human clinical trials.74The existence of high numbers of primitive hematopoietic progenitor cells in human umbilical cord blood (hUCB) has been evaluated in several studies over the last three decades. In 1974, Knudtzon demonstrated the presence of these progenitor cells in circulating human cord blood.75 Later, Broxmeyer etal. showed early experimental evidences that umbilical cord blood is a rich source of hematopoietic stem cells (HSC). However, it was only after 1989, that experimental and clinical studies were published indicating the application of human UCB in clinical translation. Soon after that Gluckman etal. first reported the hematopoietic cell transplant using UCB instead of bone marrow as the source of HSCs. They could reconstitute the hematopoietic system of a five years old child with Fanconi anemia by means of UCB from an HLA-matched sibling.77 Since then, the interest in the use of UCB as an alternate source of HSC has expanded phenomenally for both clinical and research applications.
Subsequently, numerous transplants have been performed using UCB from related and unrelated donors into not only adult patients but pediatric cases as well. UCB has the advantages of easy collection, has no risk to donors, has reduced the risks of transmitting infections, immediate availability of cryopreserved units, and acceptable partial HLA mismatches because UCB allows immunologic permissiveness.78 Advantages of UCB as HSC source for allogeneic transplantation are listed below:
Umbilical cord blood is an abundant and easily available source of stem cells, which can be harvested without any risk to the mother or infant.
Since collection occurs after birth of a full term normal infant, UCB is not associated with current ethical concerns raised in use of embryonic stem cells.
Ethnic balance in a cord blood repository can be maintained automatically in heterogeneous populations or can be controlled via collection from birthing centers representing targeted minority populations.
There is low viral contamination of UCB including cytomegalovirus and Epstein-Barr virus.
UCB, cryopreserved and banked, is easily available on demand particularly for patients with unstable disease, eliminating the complications of delay and uncertainties of marrow collection from unrelated donors.
To date, no malignant transformation of infused UCB has been observed in any transplant recipient.
The amplification of allogeneic responses including Th1-associated cytokine production by neonatal T lymphocytes has been shown to be less than that of adult T cells, which may underlie UCB reduced graft-versus-host reactivity compared with adult-derived marrow grafts.78
Several works on rodents also suggest that UCB derived stem cells have the regenerative potential to ameliorate many neurodegenerative diseases. In a rat model of spinal cord injury induced by compressing the spinal cord, when treated with human UCB leukocytes, reversal of the behavioral effects were observed even when infused 5 days after injury. It is also suggested that cord blood-derived stem cells migrate to and participate in the healing of neurological defects caused by traumatic assault.79 Taguchi etal. showed that administration of CD34+ cells from human UCB enhances neurogenesis via angiogenesis in a stroke mouse model. They have demonstrated that endogenous neurogenesis, suppressed by an antiangiogenic agent, is accelerated as a result of enhanced migration of neuronal progenitor cells to the damaged area, followed by their maturation and functional recovery.35
There are evidences that hUCB-derived MSCs secrete several immunomodulatory and neurotrophic factors, including TGFbeta-1, CNTF, NT-3 and BDNF, which are likely to play a role in neuroprotection. Further, when these cells were transplanted in a rat optic tract model, they contribute towards neural repair through rescue and regeneration of injured neurons.81 Studies have shown that intravenously delivered hUCB cells in a neonatal hypoxic-ischaemic (HI) injured rat model could improve motor functions mediated by elevating the levels of GDNF, NGF and BDNF in brain tissues. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor-mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement.82 In a canine thromboembolic brain ischemia model also hUCB-derived MSCs reduced the infarction lesion volume and differentiated into neurons and astrocytes and expressed neuroprotective factors, such as brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF), 4 weeks after transplantation.83 When seen in the context of retinal degeneration, UCB transplantations have shown hope. There have been reports showing that these cells differentiate into retinal nerve cells.84 There have been studies demonstrating efficacy of human UCB derived cells in preserving photoreceptor integrity and visual functions after injection into the subretinal space of the RCS rats.85 Some co-existing investigations had shown the failure of these cells to differentiate into any neuronal lineage after intravitreal administration in-vivo.86 It has been shown thatÂ cordÂ bloodÂ contains multipotentÂ lineage-negative SCs capable of neuronal differentiation.Â The study showed that multipotent SCsÂ derivedÂ from fresh and frozenÂ cordÂ bloodÂ samples could be efficiently induced in defined serum-free medium toward neuronal progenitors. During neuronal differentiation, the multipotent SCs underwent precise sequential changes at the molecular and cellular levels: upregulation of Ngn1, NeuN, and PSD95 and downregulation of Oct4 and Sox2, similar to neurogenesis in vivo.87 This needs further investigations for these cells to be employed in retinal repair. The UCB derived stem cells have shown expansion and differentiation both in-vitro88 and in-vivo89 conditions. There have been recent studies showing the therapeutic effects of these cells transplanted into animal models of injury and chronic diseases.89, 90, 91Since the 1980s, haematopoietic stem cell (HSC) transplantation and clinical research have mainly focused around the cell surface proteins on these cells. CD34 was one of the major cell surface markers for positive selection of heterogeneous haematopoietic stem cell population.92 CD133 was reported as another alternative marker for positive selection of this multipotent population.93 Recently the focus has shifted to another type of primitive population of lineage negative (lin-ve) stem cells in the cord blood having long term in vivo haematopoiesis potential.94 The isolation of highly purified subsets of HSCs not expressing surface antigens associated with lineage commitment (or lin- cells) has demonstrated a previously unrecognized heterogeneity within the human HSC compartment.95 Forraz etal. demonstrated the rapid isolation method of lineage negative cells from UCB mononucleated cells without expressing haematopoietic markers viz. CD45, glycophorin-A, CD38, CD7, CD33, CD56, CD16, CD3 and CD2. This primitive lin- cell population encompassed CD34+/- and CD133+/- HSCs and was also enriched for surface markers like CD164, CD162, and CXCR4 involved in HSC migration, adhesion and homing to the bone marrow.96 In another finding, CD34-CD133- CD7-CD45dimlineage (lin)- HSCs were isolated from human UCB with low but measurable extended long-term culture-initiating cell activity. These cells showed the in vitro differentiation of CFU-granulocyte-macrophage, burst-forming unit erythroid, and megakaryocytic aggregates.97 The quest for the transplantation of UCB derived stem cells has come up with the strategies involving extensive purification and characterization of these cells for isolating the most suitable population for clinical transplantation. The translational studies aim for an ethics-free, easily available and safe source of stem cells for transplantation and expansion in-vivo. A recent study suggests that theÂ cellÂ fraction containing greater than 98% CD34+ CD38-Â cellsÂ could be the ideal one for large-scale ex vivo expansion; however, the data suggested that except for MNCs, all otherÂ cellÂ populations could also be used as inputÂ cellÂ fractions. A zone of proliferating retinal stem cells exists at the far peripheral edge of the retina of fish, frogs and birds. This zone of stem cells has been termed the ciliary or circumferential marginal zone (CMZ). Mammals are known to lack CMZ, which is a potential stem source in lower vertebrates. The only mammal to have a transient CMZ is postnatal marsupials.99 A decade ago, for the first time Ahmad etal and Tropepe etal showed the existence of stem cells in the pigmented ciliary epithelium of mammals.100,101 Since then, a lot of work has been done on ciliary epithelial stem cells including rodent, rabbit, porcine, bovine, monkey and humans.102-104 These investigations gave hope for transplantation in retinal degenerative diseases. CE stem cells showed increasing amount of proliferation, when CE cells were grown in serum free media (SFM) containing mitogens like EGF, bFGF, VEGF and heparin. However, a few of pigmented epithelial cells i.e., ~ 10,000 cells/ human eye, were able to proliferate in in vitro conditions.105 It has been proposed that, the production of rod photoreceptors and other retinal cell types from CE stem cells might occur through a process of transdifferentiation. During transdifferentiation process, a stem cell with defined set of specialized progeny can give rise to different progeny106-107 by genomic reprogramming.
There are several intrinsic and extrinsic factors which affect the fate of cell. External factors like EGF, bFGF, aFGF, TGFÎ±, LIF, retinoic acid and Shh influence cell fate decisions in vitro and in vivo. Along with these factors, the microenvironment or niche plays an important role in the maintenance of the stem cells present in the pigmented ciliary epithelium. Ciliary marginal zone (CMZ) of zebrafish was known to have a close association with basal lamina surrounding blood vessels and shares several features in common with the neurogenic niche in the adult mammalian brain. Stem cell niche of CMZ germinal zone have activated Notch signaling, diffuse distribution of N cadherin, and expression of retinal homeobox genes rx1, pax6a, and vsx2/Chx10. The mammalian ciliary epithelium is involved in the production of aqueous humor and has close association with blood vasculature. The factors in close apposition to ciliary epithelium especially Vascular Endothelial Growth Factor (VEGF) may have an important role in the maintenance of stem cells present in the pigmented epithelium. VEGF was considered to be a strong angiogenic factor along with epidermal growth factor (EGF) and Fibroblast Growth Factor (FGF), however, their neurotrophic and neurogenic role has remained neglected. We therefore examined the effect of VEGF, EGF and FGF on proliferation of ciliary epithelial stem cells and demonstrated that the expression of Notch, Jagged, ï¢ catenin and N cadherin is indeed altered by these factors.
The ciliary epithelium upon induction by growth factors yields neurospheres in-vitro which on further culturing can yield cells of neural origin and probable photoreceptors. There have been studies thrashing the concept of any regenerative or differential capacity in these cells.108 Some groups have reported them to have temporary and minimal stem cell or neurogenic capacity, which is lost gradually with time.109 Surprisingly there have been co-existing groups who have reported that the same cells on culturing with differential media specific for photoreceptors for a prolonged period of time yielded viable photoreceptors that were positive by patch clamp analysis as well as calcium channel studies.110 It has been shown that clonally-derivedÂ mouse and human RSC progeny (cells derived from purification of CE) are multipotent and can differentiate into mature rhodopsin-positiveÂ cellsÂ with high efficiency using combinations of exogenous culture additives known to influence neural retinal development, including taurine and retinoic acid.110,111
According to the Neurotropic Factor Hypothesis 112 the target cells of developing neurons produce a limited amount of an essential nutrient or trophic factor that is taken up by the nerve terminals. Neurotropic factors and growth factors have been reported to stabilize retinal tissues in various degenerative retinal disorders. Photoreceptors exposed to various types of insults have been reported showing survival after the intravitreal injection of brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and basic fibroblast growth factor (bFGF), but it is not precisely known if these survival-promoting effects are induced by these factors or indirectly through the activation of surrounding retinal neurons (Wahlin etal, 2000). Therefore, it would be very useful to study the role of neurotrophic factors and how they exert their survival and differentiation promoting effects. There has been an impairment in the study of CE cells because of the lack of knowledge of cell surface markers for the characterization of these cells. The molecular mechanisms in the developmental biology of the ciliary epithelium are still not very prominent and transparent, except for the evidence of its common ancestry with the optic cup and extension of the brain ectoderm. In the recent past CD138/syndecan-1 and stage specific embryonic antigen-1 (SSEA-1)CD15 have been identified as cell-surface antigens for nonpigmented and pigmented ciliary epithelium in the developmental stages, respectively. This finding was the first to use cell-surface markers to ascertain the spatial and temporal transitions that occur in development of CE.113 The capacity of ciliary epithelium derived cells and their efficiency in getting incorporated in the degenerate retina needs to be studied and validated. The potential of these stem cells to emerge as viable source for clinical translation is probably underevaluated and needs to be validated by further studies.114
Cellular movement and migration within the central nervous system has been known to take place during development, however, it was discovered that these events occur during neurogenesis in the adult vertebrates as well. If ciliary epithelium derived stem cells are injected into subretinal space of retina, these cells are likely to migrate and get incorporated into retinal architecture. Therefore, it is necessary to map the mechanisms in these injected stem cells in order to understand which cellular signals these injected cells might use to rescue damage to retinal pigment epithelium and in altering the symptoms of retinal degeneration.Laser injury model of CNV in primates was reported in 1982,6 which has provided valuable information about the natural history of CNV, the role of the RPE in re-establishing the blood retinal barrier, and its role in the scarring process. In this model, laser burns are applied to the fundus that result in CNV but only if Bruch's membrane is disrupted. Other groups reported that laser injury alone was sufficient to induce stem cell recruitment and subsequent CNV in mice.2 Although the laser-induced model involves processes relevant to CNV, it captures many of the important features of wound healing and is, therefore, useful for studying injury responses including stem cell migration. The earlier studies on laser cast quite a good amount of light in understanding the pathophysiology of laser injuries. Laser photocoagulation that disrupts Bruch's membrane can also induce CNV in humans.115 We have modified this method in our lab by targeting the retina (sparing CNV) and causing injury by using laser of specific intensity and spot size and then study the recruitment of transplanted lin-ve BMSCs to such injury.116
Interaction of laser with biological tissue:
Laser interacts with the biological tissue through four sequential processes:
1. Transmission of laser through the ocular media
2. Absorption of laser in the target tissue
3. The conversion of laser radiation into heat energy
4. Thermal effects on the tissue being targeted
The RPE is the primary site of the photocoagulation and cellular damage as a result of laser treatment may cause release or destruction of growth factors. The laser light being an electromagnetic radiation passes through the neural retinal layers without interacting with cells in these layers as there is no pigment in these cells to interact with laser light. The RPE cells are rich in black to dark brown colored pigment melanin granules that interact with the laser light resulting in almost complete absorption of laser light at the level of RPE. The absorbed laser light gets converted into heat energy and is dissipated to the surrounding cells. This heat energy results in damage to the RPE cells, photoreceptors in the outer nuclear layer and sometimes inner nuclear layer cells are also involved in the injury. Photocoagulation is the major mechanism for laser induced damage. But secondary processes like necrosis and apoptosis are also responsible for laser injury induced cell death. The visual deterioration as a result of laser-tissue interactions is due to the nonselective tissue necrosis in the normal retina caused by the laser beam and by the thermal spread of the destructive effect to adjacent normal tissue (secondary degeneration).117-119 Currently limited treatment is available for laserÂ eye injury. In the studies involving the therapeutic potential of bone marrow-derived stem cells (BMSCs) forÂ laser-induced retinal trauma lin-ve bone marrow cells survived well after intravitreal injection.116, 120 In vivo bromodeoxyuridine (BrdU) labelling showed these cells continued to proliferate and integrate intoÂ injuredÂ retinas.120Â The studies involving the study of neurogenic inflammation, migration of microglial cells at the injury sites.121 In the transplantation experiments of different sources of stem cells following retinal photocoagulation, the enhanced level of integration of grafted BMSCs,116, 120 RPCs,122 CFSE labeled EPCs,123 is partially associated with increased expression of MMP-2 and, to a lesser extent, MMP-9, together with decreased levels of inhibitory molecules.122
It is now well documented that much of the post injury tissue damage results from delayed autodestructive mechanisms involving the formation of reactive oxygen intermediates, release of proinflammatory substances and the accumulation of intracellular calcium ion. All these mechanisms presumably lead to one final common pathway of cell death via the apoptotic cascade. Proliferation of RPE cells at site within and adjacent to sites of laser treated areas as a part of healing process has been reported
Human umbilical cord blood (hUCB) cells have multilineage differentiation capacity, and have the ability to transdifferentiate or become nonhematopoietic cells of various tissue lineages, including neural cells. When exposed to various experimental conditions, these cells showed that their progeny could reveal properties typical of neuroectoderm derived cells.124 MusashiÂ is a neural RNA-binding protein essential for neural development and required for asymmetric cell divisions in the adult sensory organ development. In the developing CNS, mouse-Musashi-1Â protein is highly enriched in the CNS stem cell. In single-cell culture experiments the association of mouse-Musashi-1 expression with neural precursor cells complimented the generation of neurons and glia. In contrast, in fully differentiated neuronal and glial cells mouse-Musashi-1Â expression is lost. This pattern of mouse-Musashi-1Â expression is complementary to that of another mammalian neural RNA-binding protein.125 Thy-1 is a glycoprotein that labels the ganglion cells and is specific to the retinal ganglion cells.126 PKCÎ± is a marker for the bipolar cells and it labels the glial cells in retina. Immunohistochemistry of 3-day-old retinas revealed intense PKC-alpha reactivity in the inner plexiform and inner nuclear cell layers, weak reactivity in the ganglion cell layer, and few positive cells in the outer nuclear cell layer. The cellular localization of PKC-alpha in the adult retina was similar, with staining more intense than that in neonates. PKC-alpha was co-localized in some glial fibrillary acidic protein-positive cells and glutamine synthetase-positive cells in the retina.127 PKC alpha is down-regulated directly by impaired protein synthesis, and also possibly indirectly by protein consumption related to GFAP up-regulation. These results were indicated in the experiment with laser photocoagulation, that interferes with PKC-alpha-mediated inhibitory regulation of inner retinal signal transmission.128 Retinal neuron and glial cells markers include rhodopsin, parvalbumin, ciliary neurotropic factor (CNTF), glutamate transporter, SV-2. Rhodopsin is a transmembrane protein in the rod photoreceptor cell membrane in the retina. It catalyses the only light sensitive step in vision. Rhodopsin is very well documented marker for rod cells in the retina. Parvalbumin is used as a marker for labeling amacrine cells in the retina. It is a cytoplasmic calcium binding protein. CNTF is a secreted growth factor of retinal ganglion cells that is used to positively label ganglion cells. Glutamate transporter is used to label Müller glia cells in the retina. SV-2 is a synaptic protein and is used as a marker for synaptogenesis. RPE 65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro. It has been a marker extensively used for the characterization of Retinal pigment epithelium cells in-vitro as well as in-vivo. It is a marker extensively used in IHC as well as in Flow cytometry for the analysis of the RPE cells.129, 130 Vascular endothelial growth factor (VEGF) is expressed by all the layers and cells of the retina and it acts as a neurotropic factor for the maintenance of the cells. Brain Derived Neurotropic factor (BDNF) is another neurotropic factor which has differential effects on the retina. Alterations in the BDNF have reported in the reductions of dopaminergic activities in amacrine cells.131 Bcl-2 and Caspase-3 are common markers extensively used for the study of apoptosis in cells. Caspases are cysteine proteases that mediate apoptosis by proteolysis of specific substrates. The Bcl-2 protein, which inhibits apoptosis, is cleaved at Asp-34 by caspases during apoptosis and by recombinant caspase-3 in vitro.Â The specific cleavage of Bcl-2 needs activation of caspase-3. Caspase-3-dependent cleavage of Bcl-2 probably promotes further caspase activation as a part of a positive feedback loop for executing the cell.132 Ki-67 is a marker associated with cell proliferation. The expression of human Ki-67 protein is strictly associated with cell proliferation. It can be detected exclusively within the nucleus during interphase, whereas in mitosis most of it is relocated to the chromosome surface. The fact that the protein Ki-67 is present in all the active phases of the cell cycle i.e.G1, S, G2, and mitosis, but is absent from resting cells i.e.G0 phase, makes it an excellent marker for determining the growth of a given cell population. Although the Ki-67 protein is extensively used as a proliferation marker and well characterized on the molecular level, the functional significance is still unclear.133 However, it is indicated that Ki-67 protein expression is absolutely required for progression of the cell through the division cycle.
To compare the efficacy of human umbilical cord blood derived lineage negative stem cells and human fetal pigmented ciliary epithelium derived neurospheres in laser injured mouse retina model.
To establish mouse model of retinal injury by laser and validate the laser spots by histology and fluorescein angiography.
To isolate, enrich and characterize the lineage negative stem cell populations in human umbilical cord blood (UCB).
Isolation, characterization and expansion of the fetal ciliary epithelium into neurospheres under the required culture conditions.
To attempt rescue of damage to retinal cells from laser by subretinal administration of lin-ve UCB derived stem cells and dissociated neurospheres from the cultured ciliary epithelium cells.
To compare stem cell migration, differentiation, integration and survival of transplanted UCB derived cells and CE derived neurospheres into retinal architecture by quantifying the expression of various stem cell markers and retinal cell markers.
To study the expression of neurotropic factors and pro & anti-apoptotic factors in laser injured retina after transplantation of stem cells with reference to other groups.
To assess the restoration of vision by ERG.
Age and sex matched syngenic C57BL/6J mice would be broadly divided into six groups. These groups will constitute the (1) control group, (2) Lin-ve UCB derived cells in injured group, (3) CE derived dissociated neurospheres in injured group, (4) Lin-ve UCB derived cells in uninjured group, (5) CE derived dissociated neurospheres in uninjured group and (6) the sham surgery group for the subretinal injection. The laser injury groups (Group 2 & 3) will be injected with stem cells subretinally 12 hrs after laser injury in both the eyes and PBS in the contralateral eye. The other two groups (Group 4 and 5) will include individual transplantation of cells from each source, the contralateral control eye injected with the diluents (media/DPBS) alone, used for cell suspension.
Each animal from these groups would be sacrificed at five time points (1st week, 2nd week, 3rd week, and 8th week). The eyes would be enucleated and processed for immunohistochemistry and Real time PCR analysis for various neuronal stem cell markers (Musashi-1), retinal cell markers [Parvalbumin, Thy-1, Synaptic vesicle (SV2A), Glutamate transporter, Rhodpsin, Rpe65, PKCÎ±] and neurotropic factors (BDNF, CNTF, VEGF), and anti- and pro-apoptotic factors (Bcl-2, caspase-3) by colocalization. The eyes would also be examined by angiography and validation of the vision loss would be carried out by ERG before sacrificing the animals.
Experiments will be performed using C57BL/6J syngenic mice after obtaining approval from Institutional Animal Ethics Committee as well as from Institutional Committee on Stem cell Research and Therapy (IC-SCRT). All efforts will be made to minimize the number of animals used and their suffering. Animals will be maintained in a 12-h light/dark cycle (LD 12:12). Animals would be fed on standard diet and would have free access to drinking water. The human samples (both CE and UCB) to be used in the study will be collected after the complete information and proper consents from the patients. The criteria for the selection of either tissue is described as follows:
Selection Criteria for Umbilical Cord Blood Donors:
Human umbilical cord blood (UCB) will be collected from the umbilical cord of newborn deliveries from pregnant women (aged between 20-35 years) at gestation period of ï‚³ 28 weeks. Proper informed consent will be filled for every donor and all the detailed procedures and objectives related to sample collection will be explained to them. All the donors will be screened and the following conditions will be excluded before collection of UCB samples:
Hepatitis B infection
Untreated urinary tract infection
Unclean vaginal examination
Prolonged rupture of membrane (> 24hrs)
Foul smelling amniotic fluid
Major congenital malformation in the new born
Selection Criteria for Ciliary Epithelium Donors:
Eyes will be obtained from abortus after legal termination of pregnancy upto 20 weeks of gestation. All the procedures will be done according to the IC-SCRT. Consent will be taken from the mother and father of abortus before obtaining eye balls. The inclusion and exclusion criterion for the collection of abortus is as following:
From mid trimester abortions
(upto 20 weeks).
Hepatitis virus B & C
Human immunodeficiency virus
Samples bearing congenital abnormalities
Any malformation of fetus effecting head
Evidence of chorioammniotisis (fever, foul smell liquor)
Intra uterine fetal death.
Argon green laser (532 nm, Iris Medical, USA) will be used to induce injury in mouse retina. Briefly, animals would be anesthetized with a cocktail of xylazine (10 mg/Kg) and ketamine (100 mg/Kg) and placed on a heating pad to prevent cold cataract. Pupils would be dilated with 1% Tropicamide solution and lignocaine solution (2%) would be applied as local anesthetic to the cornea. Anesthetized animal would be placed in front of laser photo-coagulator and a glass cover slip would be used as a lens to visualize the fundus of `mouse eye through a slit lamp. Laser would be imparted to produce 8 even spots (at a distance of around 2 optic disk diameter from optic nerve) in a circular fashion around the optic nerve with following standardized parameters:
Power of laser = 200 mW
Laser spot size = 100 microns
Duration of exposure to laser (pulse) = 100 msec
Number of spots: 8 spots per eye (around the optic disc)
During the whole procedure of laser injury, animal eyes would be kept wet by application of artificial tears. After the laser injury, Neosporin ointment would be applied to the eyes of animals to prevent infection and drying of eyes during recovery from anesthesia.
Schematic representation of laser induced injury model.
A) Front view of fundus while delivering laser injury.
B) Side view of retina being targeted with laser.
After the laser injury, animals will be sacrificed at various time points (1, 2, 3 and 8 weeks) and injured regions in retina will be identified histologically and by TUNEL assay.
Fluorescein angiography will be performed after laser injury to visualize the laser spots. The fundus of laser injured mouse eyes will be examined to observe fluorescein dye leakage and images will be captured with a fundus camera.
Mice would be sacrificed at various time points with an overdose of anesthesia and eyes will be enucleated and stored frozen in -80°C. The eyeballs will be embedded in OCT medium and 6 microns sections will be obtained. The whole eye balls will be sectioned serially in order to locate the laser injury spots and CFDA-SE labeled cells transplanted in the injured retina. After fixation with Histochoice tissue fixative, Haematoxylin and Eosin staining will be performed to identify laser spots on sections and transplanted cells.
Human umbilical cord blood (UCB) will be collected post delivery from the umbilical cord and placental blood vessels using sterile syringes in a sterile container with EDTA (anticoagulant). The UCB will be enriched by density gradient using Ficol for the nucleated cell population (NCP). This NCP will be used to isolate the Lin- stem cell population using cell surface marker based Magnetic associated cell sorter (MACS) and will be further characterized by flow cytometry for the absence of Lineage markers.
Immediately after enucleation, eyes will be transported in a sterile ice cold Hanks' Balanced Salt Solution (HBSS). Eyes will be dissected under sterile conditions using dissection microscope. Briefly, eyes will be placed in 30mm petri dish containing ice cold HBSS and cornea and lens will be removed. Eye globes will be cut in two equal halves, vitreous and retina removed. A strip of ocular tissue containing CE will be obtained by cutting the anterior edge of the pars plana. Ciliary rings will be collected and washed with HBSS. Precautions will be taken not to contaminate the tissue with non pigmented epithelium, RPE, iris and retina. For dissociation of CE tissue by trypsinization, the ciliary rings will be transferred to 15ml tube containing 4ml - 5ml of 0.25% trypsin with EDTA and incubated at 37°C water bath for 20-30 min, with shaking after every 10min. Once tissue gets digested, trypsin will be immediately neutralized with equal quantities of DMEM/F12. Cell suspension will be filtered through 0.70Î¼m cell strainer (BD biosciences) to remove unwanted debris and centrifuged at 800g for 10min. The cells will be washed thrice or more with DMEM/F12 at 800g for 10min each until the pigment washed off. Last washing will be done with retinal culture medium containing DNase1. Finally, cell pellet will be suspended in retinal culture medium (RCM) containing DMEM/F12, N2 supplement, 2mM L- glutamine, 100 U penicillin-streptomycin and fungizone. Pigmented cells will be counted either with hemocytometer or automatic cell counter (Millipore). Dissociated pigmented ciliary epithelial cells will be plated in 96 well plate with a density of 3,000 cells/well. These cells will be grown in the presence of proliferation conditions in retinal culture medium containing the following combination of growth factors i.e rhEGF (20ng/ml; R&D systems, USA), rhFGF basic (20ng/ml; R&D systems, USA and rhVEGF 165 (50ng/ml; R&D systems, USA) in CO2 incubator at 37°C. The proliferating cells i.e., neurospheres will be collected for RNA isolation and immunocytochemistry for studying the neurosphere markers. The cells from the 6th day of culture would be harvested.
CFDA-SE labeling of CFDA-SE (Vybrant CFDA cell tracer kit, Invitrogen, USA) will be used to track the recruitment of transplanted cells in laser injured retina. CFDA-SE passively diffuses into cells. It is colorless and nonfluorescent until its acetate groups are cleaved by intracellular esterases to yield highly fluorescent, amine-reactive carboxyfluorescein succinimidyl ester which reacts with intracellular amines, forming fluorescent conjugates that are well-retained inside cells. Excess unconjugated reagent and by-products passively diffuse to the extracellular medium, where they can be washed away.
Briefly, 2 ml cell suspension will be added to 2 ml of CFDA-SE solution (final conc. of CFDA in cell suspension should be between 5-10 ÂµM). Cells from both the sources will be incubated for 15 min at 37Â°C in CO2 incubator. Excess dye will be excluded from the cell suspension by incubating cells in fresh medium for another 30 minutes. Washing will be given with PBS. Cells will then be counted with a Hemocytometer/cell counter. Analysis of CFDA dye incorporated into donor cells would be performed by FACS analysis.
In vivo transplantation of cells:
The purified dissociated CE neurospheres (50,000 cells per eye/animal) derived from 6th day culture and the UCB derived lineage negative stem cells (50,000 cells per eye/animal) would be injected sub-retinally in the laser injured as well as uninjured mice as per the group. As a control, PBS would be injected in the contralateral eye of the same animal with laser injured retina. In the uninjured groups diluents alone (media/DPBS) used for cell suspension would be injected in the control eyes.
All injections would be given to anaesthetized mice and body temperature of the animals will be regulated by placing them on a heating pad to avoid cold cataract in eyes. The animals will be sacrificed at different time points to study regeneration of damaged retina by RT-PCR and IHC.
The eye sections would be observed under Fluorescent/Confocal Microscope and images captured for the transplanted cells in the sections. The fluorescent cells (CFDA labeled) would be counted manually by a double blinded study. The results will be expressed as the Mean Â±SE of the observed readings. The number of cells transplanted per unit surface area of the retina would be calculated. Mice would be sacrificed with an overdose of anesthesia (xylazine+ketamine cocktail). Eyes would be enucleated and fixed for 4 hrs (at 4oC) in 4 % paraformaldehyde (in 0.1M phosphate buffer) and freeze mounted on OCT and cryosectioned. The posterior of the eye would be sectioned sagitally, and immunohistochemistry would be performed. Briefly, sections would be incubated overnight with fluorescent or enzyme labeled antibodies against various neuronal stem cell markers (Musashi-1), Retinal and RPE cell markers (Parvalbumin, Thy-1, Synaptic vesicle (SV2A), Glutamate transporter, Rhodopsin, Rpe65, PKCÎ±, Ki67) and neurotrophic factors (VEGF, CNTF, BDNF). The sections would be examined under a fluorescent/confocal microscope to enable colocalization. Total RNA would be isolated from retinal tissue using commercially available kit and treated with RNase free DNase I. RNA would be quantified by optical density method. cDNA will then be synthesized using commercially available kit. mRNA of various markers spelled out in the study table would be quantified by real time analysis.
Apoptotic cells in the cryosections of laser injured retina will be identified by the TUNEL assay. Donor cells will also be analyzed for apoptosis after transplantation. Immunohistochemistry for the apoptotic markers (Caspase, Bcl2) would be done for analyzing the apoptosis. Fluorojade staining would also be performed on the cryosections of laser injured retina to identify the degenerating neurons as a result of injury in various study groups.
To analyze the functional outcome of retinal injury, electroretinogram (ERG) will be performed in both the laser injured and contra-lateral control eyes before injury (baseline) at different time points (i.e. 1, 2, 3 and 8 weeks) post injury using ERG unit after preliminary 24 hr darkness adaptation.
The effects of treatment upon a-wave and b-wave will be assessed by dividing the amplitude measured in stem cells transplanted group in either source of cells, by the corresponding value of the control. All amplitudes will be normalized to baseline values and will be expressed as a percent of baseline.Statistical analyses will be done by SPSS. Continuous data will be expressed as mean Â± SD. Differences between groups will be analyzed by t-test, analysis of variance, chi-square tests or Fischer exact test where appropriate.