Chronic low back pain is a major cause of disability and health care costs. Current treatments are inadequate for many patients. A number of preclinical models have been developed that attempt to mimic aspects of clinical conditions that contribute to low back pain. These involve application of nucleus pulposus material near the lumbar dorsal root ganglia (DRG), chronic compression of the DRG, or localized inflammation of the DRG. These models, which are primarily implemented in rats, have many common features including behavioral hypersensitivity of the hindpaw, enhanced excitability and spontaneous activity of sensory neurons, and locally elevated levels of inflammatory mediators including cytokines. Clinically, epidural injection of steroids (glucocorticoids) is commonly used when more conservative treatments fail, but clinical trials evaluating these treatments have yielded mixed results. There are relatively few preclinical studies of steroid effects in low back pain models. One preclinical study suggests that the mineralocorticoid receptor, also present in the DRG, may have pro-inflammatory effects that oppose the activation of the glucocorticoid receptor. Although the glucocorticoid receptor is the target of anti-inflammatory steroids, many clinically used steroids activate both receptors. This could be one explanation for the limited effects of epidural steroids in some patients. Additional preclinical research is needed to address other possible reasons for limited efficacy of steroids, such as central sensitization or presence of an ongoing inflammatory stimulus in some forms of low back pain. Low back pain is a common ailment which virtually everyone experiences at some point. In 30% of cases low back pain becomes chronic. This is a major cause of disability and an important driver of health care costs in the United States and other countries. With current therapies many patients fail to achieve adequate relief for chronic pain conditions including chronic low back pain. Causes of low back pain can include problems with the lumbar intervertebral discs, such as disc herniation, displacement or degeneration; compression of nerve roots as may occur with spinal stenosis; and inflammatory conditions such as arthritis. In the majority of cases of chronic low back pain the etiology is unknown, however -5.A number of preclinical models have been developed that attempt to mimic some of the above known causes of low back pain. Because a ruptured disc can expose the nearby dorsal root ganglion and its nerve roots to nucleus pulposus (NP), an early model of low back pain involved applying NP to the lumbar dorsal root ganglia (DRG) and/or adjacent nerve roots. This was implemented in pigs  and lateAll of the above types of back pain models implemented in rodents show increased mechanical sensitivity in the ipsilateral hind paw as measured with von Frey filaments, and many also show increased sensitivity of the paw to hot or cold thermal stimuli. The duration of this hypersensitivity varies considerably between models but can be very long-lasting; for example, local inflammation of the DRG with zymosan (LID) caused mechanical hypersensitivity lasting over 40 days .Some recent studies have called into question the relevance of reflexive measures such as the von Frey test or thermal threshold tests to understanding clinical chronic pain states . Regardiv1.6 sodium channel isoform at the time of local DRG inflammation completely blocked both mechanical hypersensitivity and abnormal spontaneous activity in myelinated cells, while having minimal effects on normal action potentials or behavior in normal animals [Several studies have examined changes in properties of the sensory neurons in compressed (CCD model) or inflamed (LID model) sensory ganglia. These two models apparently have many effects in common, including decreased rheobase and increased firing in response to injected current, as well as increased spontaneous activity or evoked bursting ,12,22. S animals . This chMost nociceptors are small diameter, unmyelinated or thinly myelinated cells. It is not yet understood how spontaneous activity, which is predominantly observed in myelinated cells, could contribute to pain behaviors in these low back pain models. One possibility is that at least some of the spontaneously active cells are myelinated nociceptors. In rat, 20% of Aαβ cells have nociceptive thresholds and response properties, and project into lamina I or II of the dorsal horn ,29. OtheInflammation has long been assumed to be a component of low back pain, which is often at least partially relieved by systemic nonsteroidal anti-inflammatory drugs or local injections of glucocorticoids (see below). In the low back pain rodent models discussed above, signs of inflammatory response in the DRG have been observed, such as activation of satellite glia cells, infiltration of macrophages, elevation of pro-inflammatory cytokines, and activation of inflammatory signaling pathways ,14,31-33The cytokines studied to date were for the most part selected on the basis of prior literature-based knowledge of individual cytokines. With the advent of techniques such as microarrays that examine essentially the entire set of genes in a rat or mouse, it is possible to examine gene expression in low back pain models without any such prior assumptions. A recent microarray study  of gene Although pain models are often classified as neuropathic or inflammatory, some experimental evidence suggests that inflammation occurring locally near the DRG has effects on the sensory neurons which are similar to those observed in neuropathic models involving axotomy near the DRG, but distinct from effects of more distant, peripheral inflammation or more distant axotomy. For example, the gene expression changes induced by local DRG inflammation in the above mentioned microarray study showed the largest overlap with those reported in microarray studies of the spinal nerve ligation model, which is commonly classified as a neuropathic pain model but which involves an injury close to the DRG; there was much less overlap with models involving more distant axotomy. Conversely, there was virtually no overlap with a microarray study of DRG gene expression changes induced by peripheral inflammation (CFA paw injection) , and theantagonist improved them [Reduction of pain behaviors by non-steroidal anti-inflammatory drugs has been demonstrated in the various rodent back pain models. Some studies used systemic application ,16, but ved them .Steroidal anti-inflammatory drugs are agonists of the glucocorticoid receptor (GR), a receptor with widespread tissue distribution which when activated has general anti-inflammatory effects, inhibiting type I inflammation  while promoting type II inflammation (tissue remodeling and wound repair). However, some clinically used steroids will also activate the mineralocorticoid receptor (MR) in vitro ,51. Thouin vitro[The possible roles of the GR and MR at the level of the spinal cord have also been examined. MR and GR are both found in the dorsal horn, and in one study using the CCD model, intrathecal GR agonists and MR antagonists could synergistically reduce thermal and mechanical pain behaviors. This suggests the two receptors have opposing actions in the spinal cord , similarin vitro. FurtherEpidural injection of glucocorticoids has become a relatively common treatment for low back pain that does not respond to more conservative treatments. However, randomized clinical trials of efficacy of epidural steroid injections for various forms of low back pain have had mixed results ,60. A re1. Might some forms of chronic back pain be due to an ongoing inflammatory process, which is not well modeled by current preclinical models? If stimuli leading to inflammation were ongoing, it might account for the failure of epidural steroid injections to provide long-lasting relief. Many studies of preclinical models of back pain are conducted at relatively short time points and may be more useful in understanding the initiation of the pain state rather than the chronic phase (which is however more relevant to patients with low back pain). It is not clear that any of the commonly used models could mimic ongoing inflammation.2. Might some of the failures of epidural steroid injections in clinical trials be due to the fact that most of the steroids clinically used for this purpose also significantly activate the MR as well as the GR, and MR activation is pro-nociceptive at the DRG level? For example, the EC50 ratios for the GR affinity/MR affinity  as measured in cell systems expressing human receptors are greater than 1 for methylprednisolone and prednisolone; for triamcinolone the ratio is 0.1 . The mos3. Might some clinical failures of epidural steroid injections be due to spinal centralization? Nociceptive inputs to the spinal cord show this form of plasticity in which a long-lasting increase in the strength of the synaptic connections in the dorsal horn can be observed following intense, repeated, and sustained nociceptor activity ,69. If sAll animal experimental procedures described herein were approved by the University of Cincinnati Institutional Animal Care and Use Committee (05-01-20-02) and were performed in accordance with the National Institutes of Health guidelines on animal care.Several preclinical models of low back pain have been developed, based on clinical conditions associated with back pain. Though differing in details and duration, these models share many similarities, including mechanical allodynia, increased excitability and spontaneous activity of sensory neurons, increased levels of pro-inflammatory cytokines, and other inflammatory changes. To date there are relatively few translational studies examining clinically used corticosteroids. Such studies might clarify the conditions under which such drugs might be effective.CCD: Chronic compression of the DRG (back pain model); CFA: Complete Freund’s adjuvant; DRG: Dorsal root ganglion; GR: Glucocorticoid receptor; IL-1β: Interleukin 1β; LID: Local inflammation of the DRG (back pain model); MCP-1: Monocyte chemoattractant protein 1; MR: Mineralocorticoid receptor; NP: Nucleus pulposus; TNF-α: Tumor necrosis factor α.JAS, WX, and J-MZ have a patent pending application for the use of eplerenone in low back pain.JAS drafted the manuscript and reviewed preclinical literature. WX helped write the manuscript and performed the experiments shown in Figures 2 and 4. FJB reviewed clinical literature and helped write the manuscript. J-MZ designed the review topic and helped write the manuscript. All authors read and approved the final manuscript.


Spinal cord injury (SCI) results in motor and sensory deficits, the severity of which depends on the level and extent of the injury. Animal models for SCI research include transection, contusion, and compression mouse models. In this paper we will discuss the endogenous stem cell response to SCI in animal models. All SCI animal models experience a similar peak of cell proliferation three days after injury; however, each specific type of injury promotes a specific and distinct stem cell response. For example, the transection model results in a strong and localized initial increase of proliferation, while in contusion and compression models, the initial level of proliferation is lower but encompasses the entire rostrocaudal extent of the spinal cord. All injury types result in an increased ependymal proliferation, but only in contusion and compression models is there a significant level of proliferation in the lateral regions of the spinal cord. Finally, the fate of newly generated cells varies from a mainly oligodendrocyte fate in contusion and compression to a mostly astrocyte fate in the transection model. Here we will discuss the potential of endogenous stem/progenitor cell manipulation as a therapeutic tool to treat SCI. In contrast to the former dogma that states that the adult mammalian central nervous system (CNS) is a tissue incapable of cell proliferation , 2, neurAn estimated 265,000 people in the United States suffer some form of SCI . LifetimSCI consists of a primary injury that leads to a secondary injury cascade. The primary injury is a physical insult, commonly induced by a compressive force of the vertebrae on the spinal cord . This meStudies on cell response to SCI in humans are very limited due to issues of patient consent, technological constraints, medical urgency, and tissue availability. Consequently, animal models for SCI form the basis for much of our current knowledge on how cells in the spinal cord are affected by injury. The most common clinical presentation of SCI in human patients is a fracture dislocation injury in which the vertebrae are compressed against the spinal cord. The contusion model of animal SCI mimics this injury by applying a force onto the dorsal aspect of the spinal cord. By using this model on laboratory rodents, we can gain a better understanding of the precise events that take place in the spinal cord after injury and how regenerative therapies may modulate cellular responses to injury. + cells labeled decreased by 67% within one day of a hemisection injury in mouse [Spinal cord injury causes rapid loss of all cell types within the spinal cord. In a model of incomplete contusion (10 g weight from a height of 2.5 cm), the rat spinal cord lost approximately 66% of the ventral motor neurons at the injury epicenter within 15 minutes of injury, and there was a near complete loss within four hours . Oligodein mouse . Approxiin mouse , a resulin mouse . The losin mouse . After iin mouse . To mimiValuable animal research models for SCI should demonstrate reproducible deficits in locomotor and sensory behaviors. Furthermore, SCI animal models should be adjustable to produce injuries of differing severity, which must correlate with the degree of neurological deficit observed. There are many forms of SCI, both clinically observed and experimentally validated, that can be classified into three groups of models based on the type of injury: transection, contusion, and compression.Transection SCI models are all forms of laceration to the spinal cord. Some common transection models include complete transection, in which a blade is passed through the entirety of the dura mater and spinal cord, dorsal or lateral hemisection, in which a blade is passed only half-way through the spinal cord , and incContusion injuries are produced by a focal force acting on the spinal cord, most commonly from the dorsal aspect . FeatureCompression injuries closely resemble contusion injuries in terms of pathological properties and neurological impairment; however, they differ in several regards. Contusion injuries are focal dorsal compressions, while in compression models the force can be applied either laterally or dorsally depending on the apparatus used. A compression injury can be generated using the Plemel method of calibrated forceps compression , aneurysin vitro [in vitro and are capable of becoming neurons when transplanted into the hippocampus, but when transplanted into the spinal cord they only acquire glial fates [in vitro capability and in vivo behavior raises the question of whether or not spinal cord cells capable of forming neurospheres in vitro are truly multipotent stem cells. In this paper we will discuss experiments used to identify populations of cells within specific regions of the spinal cord with capacity to self-renew and generate daughter cells that express markers of mature CNS cells. Neural stem cells (NSCs) are inherently difficult to identify and study. This is due in a large part to their heterogeneity of morphology and marker expression, as well as the lack of an identified anatomical niche –52. The in vitro , 58, 60.in vitro , 60. An in vivo. Later studies by Johansson et al. [Although early studies showed postnatal neurogenesis in the brain –63, studn et al.  and Yaman et al.  providedThe ependymal layer of the spinal cord has received much attention due to its role during embryonic development as a neuroprogenitor cell niche, its relation to the ependymal and subependymal layers of the brain, and its role in the reconnection and regeneration of the spinal cord of lower vertebrates. The central canal of the postnatal vertebrate spinal cord is congruent with the ventricular system of the brain and is similarly lined with a pseudostratified layer of ependymal cells , 64, 65.α), Sox3, FoxJ1, and Notch1 receptor [Ependymal cells in the mammalian spinal cord express markers associated with neural stem cells such as Nestin, GFAP, brain lipid binding protein (BLBP), Sox2, Vimentin, Musashi1, alpha-type platelet derived growth factor , and for oligodendrocytes, such as antioligodendrocyte marker clone NS-1 (RIP), but did not express the neuronal markers class III β-tubulin (Tuj1) or neuronal nuclei (NeuN). A similar study by the Frisén Laboratory, using transgenic mice with an inducible Cre-LoxP system for lineage tracing, found that only ependymal cells were able to generate neurospheres. Proliferating cells of the Olig2+ lineage in the parenchyma did not but self-renewed and generated mature oligodendrocytes, ascribing to them a role as OPCs [+ cells as a candidate cell population to fulfill the role of stem/progenitor cells observed in the lateral parenchyma by Yamamoto et al. and Horner et al. The existence of a stem/progenitor cell niche in the lateral parenchyma is further supported by a recent study by Petit et al. that proposed, based on morphology and marker expression, that some nonependymal proliferating cells located in the subpial white matter of the mouse spinal cord are putative adult radial glia (RG) [+ RG [+ cells with RG morphology are localized to the central gelatinosa surrounding the central canal in the adult turtle [+ cells do not reconnect the spinal cord but undergo mitosis at an increased rate after injury [+ cells of Petit et al. may be analogous to RG-derived astroglia in the adult brain. However, due to similarities in marker expression, morphology, and response to injury, further study is needed to differentiate this cell population from functional astrocytes. BLBP+ cells from the subpial white matter of the spinal cord could be isolated and cultured under conditions conducive to neurosphere formation to determine if they represent a pool of multipotent cells in the lateral parenchyma of the adult spinal cord. Recent studies also identified stem/progenitor cell proliferation in regions of the spinal cord away from the central canal. Horner et al. described proliferation in regions of the lateral parenchyma in intact adult rat spinal cord . By usin as OPCs . On the  as OPCs . The Yamlia (RG) . These cG) [+ RG . Additiot turtle , 75, aret turtle , 76 and t turtle . In mammr injury . Confounr injury , 78–82. + cells are capable of forming self-renewing neurospheres in vitro that are only able to produce oligodendrocytes [+ cells in the white matter are preferentially associated with radial elements of the spinal cord [+ cells are closely associated with RG cells [+ cells with radial elements in the spinal cord and the affiliation of putative adult RG cells described by Petit et al. with these same elements [+ cells during development. Ng2+ cells are generally considered to be OPCs; however, there is a large body of evidence suggesting that there are two distinct Ng2+ cell populations and that only one of them functions as an OPC [+ cells differ in morphology; one class of Ng2+ cells is stellate shaped with highly branched processes, while the other class of Ng2+ cells has a flat, epithelioid soma and fewer processes [+ cells differ in their response to injury and growth factor treatment, electrophysiology, and in their potential for acquiring specific cell fates [As the predominant dividing cell type in the intact postnatal spinal cord , Ng2 expdrocytes , expressdrocytes , 84, 85.drocytes , althougRG cells ; thus thelements  is simils an OPC , 88, 89.rocesses , 86, 89.ll fates . + cells within the outer circumference in the cervical and lumbar spinal cord than in the thoracic spinal cord [+ putative RG cells at the pial edge in a cervical to lumbar gradient [+ cells can be accounted for by BrdU labeling in the Horner study, which labeled all mitotic cells, but Olig2+ cells did not give rise to neurospheres and would not be represented in the Weiss study. If the cells described by Petit et al. are true stem cells capable of neurosphere formation, their population gradient might explain the increased numbers of neurospheres generated from lumbar spinal cord by Weiss et al. Using neurosphere formation as a criteria for defining a pool of stem/progenitor cells, these studies provide evidence that there is a cervical to lumbar gradient of stem/progenitor cells, and that specific levels of the spinal cord may have the capacity to generate a more robust and efficient response to injury. Of further note is the distribution of stem/progenitor cells throughout the spinal cord along the rostral-caudal axis. Horner et al. found more BrdUnal cord , while Pgradient . Similargradient , but it in vitro [+ cells had proliferated in response to injury [in vitro, indicating that some cells that proliferate in response to SCI are multipotent under specific conditions; however, the lack of neurogenesis after injury in vivo indicates that the postnatal spinal cord environment is not conducive to the generation of new neurons.Lower vertebrates, such as eel , axolotlin vitro , 8; howein vitro , 28. Fur+ and BLBP+ cells, was formed between the resected ends of the spinal cord [Proliferation at the central canal plays an important role in the reconnection of the transected spinal cord of lower vertebrates such as the eel , turtle nal cord . By the in vivo proliferation rate of spinal cord ependymal cells was very low, but after an incision in the dorsal funiculus, the proliferation of the ependymal layer increased 50-fold [+ cells were present within 70 μm of the central canal [μm from the central canal towards the lesion at later time points. Additional experiments on the proliferative response to a more severe transection injury by Horky et al. described a significant increase in BrdU+ cells after a dorsal hemisection injury in mouse [+ cells of the ependymal layer expressed either S100b or Nestin at early time points, which diminished over time. Within the spinal cord parenchyma, BrdU+/Ng2+ cells were abundant at all time points, while mature oligodendrocytes expressing CC1 were present one week after injury.In contrast to the significant functional recovery in lower vertebrates, the spinal cord of higher order vertebrates such as rat and mouse has a far more limited potential for spontaneous recovery of motor and sensory function after injury. However, like lower vertebrates, mammals experience cell proliferation and there is evidence of repair mechanisms in the form of the remyelination of damaged axons. One of the earliest studies to characterize mammalian ependymal cells as putative progenitor cells examined the ependymal response to incision injury in rats. The  50-fold . Ependym 50-fold , ependymal canal . Migratiin mouse . These c+ or Pax7+ cells coexpressed BrdU. Due to the timing of their emergence and distance from the ependymal layer, the authors concluded that Pax6+ cells in the lateral parenchyma could not have migrated from the central canal. It is possible that these cells were upregulating Pax6 in response to injury and/or becoming mitotic and producing daughter cells that expressed Pax6. Expression of Nkx2.2 increased throughout the medial and lateral parenchyma in a temporal pattern similar to that observed in the Pax6+ and Pax7+ cell populations. Pax6, Pax7, and Nkx2.2 are master regulators that confer a regional identity on neuroprogenitor cells during the embryonic development, with Pax6+ cells giving rise to ventral motoneurons and oligodendrocytes [+ cells giving rise to dorsal interneurons [+ cells giving rise to primarily oligodendrocytes [in vitro in a recapitulation of embryonic development, they did not observe similar gene expression in vivo after a transection injury [+ cells than neurospheres cultured without a Notch1 inhibitor. This study raises the hypothesis that Notch1 is a potential factor that contributes to limiting neurogenesis in the postnatal spinal cord and identifies Notch1 as a potential therapeutic target for regenerative therapies targeting endogenous stem/progenitor cells in the spinal cord. As already discussed, Yamamoto et al. was able to generate neurospheres from the lateral parenchyma of both intact and injured rat spinal cord . In a fodrocytes –97, Pax7rneurons , 98, 99,drocytes  and a sudrocytes . Additiodrocytes –82. Pax6drocytes , 90, 91.drocytes , 98, 99.n injury . Neurospin vivo after injury. They performed a dorsal funiculus injury in transgenic mice in which Foxj1+ ependymal cells, Cx30+ astrocytes, or Olig2+ oligodendrocytes were labeled for cell fate mapping purposes. Ependymal lineage cells increased four- to five-fold at the injury site compared to adjacent regions, while cells of the glial lineages increased twofold in response to injury [Studies from the Frisén Laboratory have attempted to address the debate of which cell population(s) function as neural stem cells o injury . Cell pro injury . Similaro injury . This mao injury , 29. + cells both within the cervical lesion as well as in unlesioned lumbar segments of the spinal cord [+ cells in the lesion were Iba1+ microglia seven weeks after injury. These Iba1+ cells could derive from peripheral macrophages that infiltrated the damaged spinal cord tissue and proliferated. The issue of nonnative cell proliferation accounting for BrdU+ cells has been raised by other researchers and addressed by Horky et al., who performed a hemisection injury on bone marrow chimeric mice. They traced the migration of GFP-labeled bone marrow stromal cells into the spinal cord after injury and determined that less than 10% of BrdU+ cells originated from proliferating peripheral blood cells [+/Iba1+ cells observed in the primate spinal cord after transection injury derived from cell infiltrates and instead may be the result of endogenous proliferating microglia or macrophages. Some proliferative cells in the macaque spinal cord also expressed Olig2, although this proportion declined over time as mature oligodendrocyte and astrocyte markers increased [+ microglial cells, features similar to reported results in animal models of contusive and compressive SCI. Proliferation after injury has also been observed in nonhuman primates (macaque) with varying degrees of transection injuries. Yang et al. described how a more severe injury correlated with increased numbers of BrdUnal cord . In the od cells . It is tncreased . There w+ cells to injury. Their experiments demonstrated changes in the post-injury extracellular environment of the spinal cord after hemisection injury and the effect that these cues had on Ng2+. To label proliferating Ng2+ cells, which are likely to be OPCs, they used an Ng2 promoter-specific retrovirus [+ astrocytes, Iba1+ microglia, and pericytes. This study produced several interesting results, such as the generation of S100b+ phagocytic astrocytes from Ng2+ cells, which appeared within 24 hours at the lesion epicenter and appeared to be engaged in clearing myelin debris. However, a significant finding was that Ng2+ cells generated at different time points after injury acquired different cell fates. Cells labeled with retrovirus on the first day after injury acquired an astrocyte fate by the third day, while cells labeled on the seventh day became oligodendrocytes expressing myelin basic protein (MBP). These changes in cell fate determination were closely linked to changes in growth factor expression within the spinal cord after injury. Sellers et al. focused on the response of Ng2trovirus . This re+ OPCs and Ng2+ non-OPCs in the parenchyma also entered a mitotic program. Although stem/progenitor cells generate a variety of cell types, the majority of newly born daughter cells acquired astrocytic fates  were labeled with enhanced green fluorescent protein (EGFP), Lytle et al. differentiated between Ng2+ OPCs and Ng2+ non-OPCs [+ cells proliferated in response to injury; however, they exhibited distinct spatiotemporal population dynamics, with Ng2+ OPCs reaching a proliferative peak three days after injury and non-OPCs reaching a peak seven days after injury. Although both Ng2+ cell types upregulated Nestin in response to injury, non-OPCs did not express CC1 at any time. Furthermore, Ng2+ OPCs followed a developmental program of oligodendrogliosis. During the acute phase of injury, Olig2 was upregulated, while Olig1 and Nkx 2.2 were downregulated. At later time points, Olig1 and Nkx2.2 were upregulated, while Olig2 expression decreased; cells that underwent such changes in their transcriptional profile ultimately became CC1+ oligodendrocytes. These findings contrast with an earlier report by Yamamoto et al. where there was no expression of these transcription factors in a rat model of transection [Yoo and Wrathall enriched Ng2trocytes , and thunon-OPCs . Both tynsection . It is mIn contrast to the stem/progenitor cell response to transection injury, a contusion injury triggers a systemic mitotic response through the entire rostrocaudal extent of the spinal cord . The cel+ cell response to compression injury resembled contusion injury [+ cells in the subpial white matter and dorsal funicles in regions five mm rostral and caudal to the injury. One week after injury, there was an increase of Ng2 immunoreactivity in the subpial white matter along with a decrease in immunoreactivity in the dorsal funicles. Many bipolar Ng2+ cells with long processes extending from the subpial region of the dorsal spinal cord colocalized with 3CB-2, a marker for RG cells. These results support the hypothesis that there is a population of stem/progenitor cells in the subpial white matter of the adult spinal cord with features of RG cells [Compression and contusion injuries are similar in their pathology and have comparable cellular responses to injury. Wu et al. found that the Ng2n injury . 24 hourRG cells , 52. + cells 24 hours after injury [+ cells were located in the ependymal layer adjacent to the lesion, with the number of labeled cells decreasing with distance from the lesion [+ cells were present. Within six hours of injury there was a little change in Nestin expression; however, 24 hours after injury there was an increase in immunoreactivity. At later time points Nestin expression continued to increase and spread beyond the ependymal layer; however, not all Nestin+ cells were simultaneously Ki-67+, providing further evidence that some stem/progenitor cells may upregulate neuronal stem cell markers but not undergo mitosis in response to injury. In minimal injury [Proliferation at the central canal has also been observed in compression models of SCI, similar to that in contusive models. An analysis of ependymal proliferation and expression of Nestin after clip compression injury reported an increase in Ki-67r injury . The eper injury , 29. Mose lesion . Althougl injury  SCI model injury . FurtherAlthough the compression model of SCI is not as popular as the contusion and transection models, they share common features such as stem/progenitor cell proliferation peaking at three days after injury. Furthermore, the pathological features of the post injury spinal cord and cell fate of daughter cells closely resemble endogenous responses to contusive injury . One of the most promising tools for functional recovery after SCI is the replacement of cells lost as a consequence of trauma. Cell replacement can be achieved by the transplantation of exogenous stem cells, such as cells derived from embryonic stem cells (ESCs) , 102, ScDespite the encouraging results from stem cell transplantation, the use of certain stem cells can raise ethical concerns, cells may be difficult to obtain in sufficient quantities for the treatment of human patients, and there are concerns about tumorigenicity and immunogenicity. Thus, manipulating endogenous stem/progenitor cells is an attractive alternative regenerative therapy for SCI. Although an endogenous mitotic program is activated in response to injury, there is a persistent tissue shrinkage indicative of a massive loss of cells. The goal of manipulating endogenous stem/progenitor cells is to increase proliferation to recuperate lost neurons, oligodendrocytes, and astrocytes. Differentiating stem/progenitor cells into neurons is a priority, as the endogenous response appears to be biased toward replacing OPCs and astrocytes rather than generating new neurons. A potential approach for manipulating the endogenous response to injury is the administration of growth factors or cytokines to modulate the milieu in which endogenous cells are proliferating and differentiating. α, which is implicated in OPC proliferation [Brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factor (CTNF), vascular endothelial growth factor (VEGF), and many other growth factors have neuroprotective and neurotrophic properties that ameliorate neuronal death after injury , 27, 117feration , and BMPferation . How besin vitro [+ cells increased after contusive SCI, as did the expression of the gliogenic factors CNTF and glial growth factor (GGF2) [+ oligodendrocytes and Ng2+ non-OPCs increased, while the total number of Ng2+ OPCs was unaffected by growth factor treatment [+ OPCs to maintain their population number despite enhanced mitosis and differentiation of their progeny. The failure of FGF2 to induce multipotency in vivo suggests that regenerative therapies may need to be more sophisticated and targeted to a number of biochemical pathways. For example, Notch1 is upregulated during the first week after injury [in vivo. In vitro data reveals that neurospheres originating from injured spinal cord and cultured in the presence of a Notch1 inhibitor generated more Tuj1+ cells than neurospheres cultured without this inhibitor. A successful combination therapy may need two or more components, such as FGF2 to increase stem/progenitor cell proliferation and Notch1 to encourage neuronal differentiation. Early reports on the isolation and culture of multipotent spinal cord progenitors indicate that EGF and/or FGF2 is necessary for neurosphere formation and the multipotential differentiation capabilities of spinal stem/progenitor cells in vitro . Howeverr (GGF2) . When GGreatment . The latr injury . Notch1 in vivo environment and induce changes in cell fate determination include the direct injection of BMP4 into a hemisected spinal cord [+ cells and CC1+ cells decreased at the injury site. Reducing astrocytes may be beneficial by reducing the occurrence of allodynia and/or reducing the amount of neuroinhibitory molecules secreted by reactive astrocytes [+ cells increased and CC1+ cells decreased. Thus, there may be conflicting cues in the spinal cord that affect the ability of stem/progenitor cells to generate specific cell types, and factors which are neurogenic during developmental processes may not have the same effect on adult stem/progenitor cells. Further evaluation of growth factor effects after SCI are needed to develop optimal therapies to promote neurogenesis in vivo. Basic research to determine how and why spinal stem/progenitor cells behave so differently in vitro and in vivo may allow for the design of regenerative therapies that increase cellular plasticity after injury. There is a wealth of in vitro data that we can draw on to develop new combinatorial therapies. For example, in vitro studies suggest that Olig2 and Mash1 enhance the differentiation of NSCs into mature neurons [in vivo should be performed to determine if they are capable of improving sensory and motor function after trauma. Other attempts to manipulate the nal cord . Sellerstrocytes ; however neurons . Other s neurons  and on a neurons . Althoug neurons , the timβ-galactosidase [Administration of growth factors, or lineage specific transcription factors, by a viral vector is another method for introducing therapeutic factors to the spinal cord. Adenoviral mediated overexpression of BDNF and Noggin in the adult striatum encouraged resident NPCs to differentiate into functional neurons , a theratosidase .Administration of growth factors can also be accomplished by transplanting stem cells that have undergone genetic modifications to express growth factors in a specific region of the spinal cord or CNS. MSCs expressing BDNF preserved a retinal and optic nerve function when transplanted into a rat model of chronic ocular hypertension , and impin vitro and in vivo stem/progenitor cell response to growth factors and transcription factor overexpression, as well as the complexity of treatment design. The response to injury is complex; thus there is need for regenerative treatments targeting more than one factor to not only change the post injury environment, but also to modulate progenitor cell fate.A number of reports published in the last decade on regenerative therapies for SCI highlight the discrepancy between the Some aspects of the mammalian response to SCI are common regardless of the injury type. However, other features, such as the location, extent, and rate of cell proliferation, vary depending on the type and severity of the injury . In most+ or Olig2+ cells. Nevertheless, taken together, the literature suggests that ependymal cells of the adult spinal cord are heterogeneous in the nature and that there are distinct stem/progenitor cell populations located within the parenchyma and ependymal layer that proliferate and generate specific spinal cord cells after injury. In addition, generated cells acquire different fates depending on injury type (+ or S100b+ cells) and contribute to the formation of a fibrous glial scar. However, in contusion and compression models the majority of new cells outside of the lesion express markers for OPCs (Ng2+) and later for mature oligodendrocytes (CC1+). Compressive or contusive injuries result in ischemic and necrotic conditions that contribute to the demyelination of spinal tracts and cell death; so the replenishment of mature myelinating cells is necessary to prevent further degradation of axons [The cell types that proliferate after injury vary between experimental injury models. However, this phenomenon may be attributed to experimental factors, such as a limited focus of each study, the selection of a transgenic approach, or the choice of immunohistochemical markers see . Some stury type . After tof axons . Prolifeof axons , there iDue to ethical issues, safety concerns, and technical obstacles in the use of human stem cells for treatment, the modulation of endogenous stem/progenitor cells in the spinal cord represents an attractive alternative to embryonic or induced pluripotent stem cell transplantation. Since the proliferation of endogenous stem/progenitor cells occurs during the first three days after injury, therapeutic approaches should target time points before the third day. Therapeutic intervention should also be considered at later time points to adjust the milieu in which the cells are differentiating and maturing. Furthermore, as there are clearly differences in the endogenous response between the different types of SCI, these therapies will need to be optimized for injury type and severity. For example, inhibitors directed against glial scar associated extracellular matrix components will be more effective in transection injuries, where a glial scar is a pathological feature which inhibits axonal sprouting. Increasing the rate of neurogenesis should be a goal for all injury types; however in contusive injuries it may be beneficial to supplement endogenous astrocyte generation, as there is evidence that specific astrocyte subtypes may aid in functional recovery after SCI , 129, al


Cardiovascular disease (CVD) is a heterogeneous, complex trait that has a major impact on human morbidity and mortality. Common genetic variation may predispose to common forms of CVD in the community, and rare genetic conditions provide unique pathogenetic insights into these diseases. With the advent of the Human Genome Project and the genomic era, new tools and methodologies have revolutionised the field of genetic research in cardiovascular medicine. In this review, we describe the rationale for the current emphasis on large-scale genomic studies, elaborate on genome wide association studies and summarise the impact of genomics on clinical cardiovascular medicine and how this may eventually lead to new therapeutics and personalised medicine. Cardiovascular disease: CVD is a heterogeneous, complex trait that has a major impact on human morbidity and mortality. The field of cardiovascular medicine involves a broad spectrum of abnormalities that are characterised by various clinical and etiological features – the major categories being coronary artery disease, congenital heart disease, heart muscle disorders and conduction disorders. Despite tremendous progress in knowledge gained, CVD remains the leading cause of death in the United Kingdom  and it hHowever, the clinical practice of cardiovascular medicine is largely governed by a phenotype based approach. Nevertheless, functional genomics has led to an improvement of our understanding of CVD and can be translated to clinical utility. Gene-based pre-symptomatic prediction of illness, finer diagnostic sub-classifications and improved risk assessment tools will permit earlier and more targeted intervention. Pharmacogenetics will guide our therapeutic decisions and monitor response to therapy. Personalised medicine requires the integration of clinical information, stable and dynamic genomics and molecular phenotyping.In this review, we describe the rationale for the current emphasis on large-scale genomic studies, elaborate on genome wide association studies and summarise the impact of genomics on clinical cardiovascular medicine and how this may eventually lead to new therapeutics and personalised medicine.We have accepted that CVD is a complex genetic trait showing familiality but no precise mode of inheritance. However, in the “pregenome era” pedigree studies have provided evidence for Mendelian mode of transmission for rare familial CVD, ion channel dysfunction underlying long QT syndrome, and sarcomeric and cytoskeletal protein abnormalities underlying hypertrophic and dilated cardiomyopathies. However, these mutations only account for a minority of the genetic basis of cardiovascular diseases -4. It isGWAS have become more popular in recent years. Their rise can be attributed to the availability of whole genome Single Nucleotide Polymorphism (SNP – variant DNA sequence due to change in a single nucleotide) panels , large-scale genotyping technologies that are available at multiple genetics centres, the rapidly dropping sequencing cost per genome and powerful association analysis methods and accompanying software , 11.GWAS are case-control studies, wherein research subjects are typed for a large number of SNPs, typically 3, 000,000 to 10,000,000, and the allele or genotype frequencies are evaluated for differences between groups or for correlations with continuous traits. GWAS are primarily designed to provide an unbiased survey of the effects of common genetic variants. The power of the GWAS to detect the phenotype associated alleles depends directly on the sample size of the study population, MAF , strength of LD between the markers, the causal variants and the effect sizes of the alleles.P-value at the genome-wide significance level (P< 5 x 10-8) and (3) confirmation of the association by independent replication studies [At present, 3 conditions must be satisfied to be considered susceptible loci through GWAS: (1) sufficient sample size in the genome-wide scan (at least 1000 each of cases and controls), (2) association  studies .P < 5 × 10−8 and confirmed the association of 10 of 12 previously reported CAD loci. The 13 new loci showed risk allele frequencies ranging from 0.13 to 0.91 and were associated with a 6% to 17% increase in the risk of CAD per allele [-3. Subsequent use of data on genetic variants and addition of data on global monocytic gene expression revealed a novel association signal at chromosome 10q 23.31 within the LIPA: lysosomal lipase acid A gene. An assessment of LIPA SNPs and transcript with cardiovascular phenotype revealed an association of LIPA transcript levels with impaired endothelial function [1).It has been estimated that heritable factors account for 30%–60% of the inter-individual variation in the risk of coronary artery disease: CAD . Genome-r allele . A recenfunction . The GWAThe QT interval represents the time for both ventricular depolarisation and repolarisation to occur and hence estimates the duration of ventricular action potential. Long QT syndrome is due to prolonged repolarisation of the ventricular myocyte, manifested as a prolonged QT interval on the ECG that predisposes to ventricular tachycardia in the form of torsade des pointes, ventricular fibrillation and sudden cardiac death. Long QT syndrome is typically an isolated, autosomal dominant condition with significant variability in disease penetrance even in those harbouring the same disease-causing mutation, although the condition may be associated with sensorineural deafness and a more severe cardiac phenotype when inherited in a recessive fashion. However, the genetic mutations identified in the Mendelian form explain < 25% of the heritability. NOS1AP gene (1q23.3) in a staged study that involved 2 independent cohorts and was subsequently independently replicated in another large cohort [NOS1AP does not encode an ion channel but rather is a regulator of nitric oxide synthase; its mechanism of prolonging the QT interval is unknown. Moreover, 9 additional loci along-with NOS1AP have been associated with QT duration by the QTSCD and QTGEN consortium [QT-interval length on ECG was among the first CVD phenotypes to be studied by GWAS, revealing a strong association with SNPs in the e cohort , 25. Unlnsortium , 27, whiThe electrocardiographic QRS interval reflects ventricular depolarisation, and its duration is a function of electrophysiological properties within the His-Purkinje system and the ventricular myocardium. A diseased ventricular conduction system can lead to life-threatening brady-arrhythmias, such as heart block, and death in the general population and in cohorts with hypertension and coronary artery disease -30. In aTwin and family studies suggest a genetic contribution to QRS duration, with heritability estimates of up to 40% , 33. CanSCN5A-SCN10A; locus 2, CDKN1A; locus 8, TBX5; and locus 21, DKK1) were previously associated with QRS duration in smaller independent studies using both candidate gene and genome-wide approaches [SCN10A, a candidate gene at the most significantly associated locus in this study, is expressed in the mouse ventricular conduction system, and treatment with a selective SCN10A blocker prolongs QRS duration. GWAS loci associated with QRS duration are illustrated in (Table 2).A recent meta-analysis of 14 GWAS consisting of 40,407 individuals of European descent with additional genotyping in 7170 Europeans yielded genome wide significance associations of QRS duration with common variants in 22 loci. Variation in four of these loci  as well as atrial and ventricular fibrillation [It has also been shown in previous studies that in addition to their association with QRS duration, variants in illation , 36, 38.Stroke is the leading cause of morbidity and mortality in the developed world. It can be broadly classified into ischaemic  and haemorrhagic stroke; with ischaemic stroke responsible for >80% of the cerebrovascular events.In the first successful GWAS for ischaemic stroke  the onlyth of stroke cases.Bilguvar and colleagues  identifiGiven the complex aetiology of CVD, the study of cardiovascular risk factors is likely to facilitate the elucidation of cardiovascular risk. Association studies have targeted multiple unrelated phenotypes measured in epidemiological surveys. Given that effect-sizes for variants identified through GWAS were smaller than previously suspected, many groups have now combined GWAS scans to identify novel loci for these quantitative traits , 48-52.In two co-published papers, analysis of lipid concentrations in a combined total of 8 816 individuals  with folSLC30A8, HHEX, LOC387761, conand EXT2 and validated the previously identified association at TCF7L2 [To date, GWAS have identified nearly 40 susceptibility loci for type 2 diabetes: T2D in European and Asian populations. The first GWAS for T2D was conducted in a French cohort composed of 661 cases and 614 controls, covering 392,935 SNP loci. This study identified novel association signals at t TCF7L2 .TCF7L2, SLC30A8, HHEX, CDKAL1, IGF2BP2, CDKN2A/B, PPARG, and KCNJ11. In addition to these 8 loci, the WTCCC/UKT2D study identified a strong association between FTO variants and T2D, although the effect of FTO variants on conferring susceptibility to T2D was mostly mediated through increase in body weight [The first round of European GWAS confirmed 8 T2D susceptibility loci across multiple ethnic groups: y weight . Most ofy weight .A GWAS of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent identified 16 novel loci: six of these contain genes previously known or suspected to regulate blood pressure  and the other ten provide new clues to blood pressure physiology .-5 were then followed up in two separate sporadic TAAD cohorts.The associated SNPs were found in a large region in linkage disequilibrium encompassing FBN1, which encodes fibrillin. FBN1 mutations cause Marfan syndrome, whose major cardiovascular complication is sporadic TAAD. The study suggested that common genetic variants at 15q 21.1, (that probably act via FBN1) are associated with sporadic TAAD, suggesting a common pathogenesis of aortic disease in Marfan syndrome and sporadic TAAD [Thoracic aortic aneurysms and dissections: TAAD can be inherited as a single gene disorder, but the genetic predisposition in majority of the people is poorly understood. A recent multi-staged GWAS, compared 765 individuals with sporadic TAAD with 874 controls. The study identified common SNPs at 15q21.1 locus that associated with sporadic TAAD and attained genome wide significance. 107 SNPs associated with sporadic TAAD with p < 1x 10 dic TAAD .Although GWAS have been very useful in identifying a large number of phenotype associated alleles, that may lead to novel pathways involved in the pathogenesis of the phenotype, they do have some rather important limitations. The results of GWAS are subject to multiple-hypothesis testing. This is because a very large number of SNPs are typed to look for possible associations and hence this needs to be corrected for the possibility of random associations in statistical analysis. In addition, often the same study population is analysed for the association of the genotypes with multiple phenotypes, which also increases the likelihood of spurious associations. Despite the very large number of phenotype- associated alleles identified by GWAS, the number of functional SNP associated alleles is scarce . Hence tAlleles identified in GWAS are often not the true causative alleles but are likely in linkage disequilibrium with the true causative alleles. The precise identification of gene and causal variants in complex diseases is difficult. The most challenging aspect is that most associated SNPs map to non-coding segments of the genome; although these regions contribute to complex diseases they contain genetic motifs that we do not fully recognise, and thus do not necessarily identify a specific gene . Targeterd Generation” sequencing technology (see below).The results of GWAS have minimal to modest impact, if any, on preclinical diagnosis, risk stratification, or genetic based prevention and treatment at an individual level. This could be due to large number of common variants with low magnitude of effect, rare variants with large effects, interaction between alleles at homologous loci and between alleles at non-homologous loci, epigenetic effects and underestimation of the effect of shared environment among relatives leading to inflated estimation of heritability . Also, tBy their very nature, GWAS focus on a very small percentage of the total genome, thus there is great potential to miss the causative variations, irrespective of whether these are in coding or non-coding regions. However, capturing all possible variation within a sample requires a sequencing strategy. Since first introduced to the market in 2005, next-generation sequencing technologies have had a tremendous impact on genomic research. The next-generation technologies have been used for standard sequencing applications, such as genome sequencing and re-sequencing, and for novel applications previously unexplored by Sanger sequencing. The landmark publications of the late 1970 s by Sanger's and Gilbert's groups and notably the development of the chain termination method by Sanger and colleagues  establisnd Generation” sequencing essentially relies on a modified “shotgun” approach, involving multiple steps: template fragmentation and amplification, sequencing, imaging and alignment of the sequences to a reference genome. A key aspect is that current technology is unable to sequence much more that 1000 base pairs in a single reaction; indeed much of the published data is derived from very short sequencing “reads” of around 35bp, something that can cause problems with downstream analysis, as these short reads may align to multiple locations in the reference genome requiring computationally expensive alignment algorithms. Artefacts can also be introduced at various steps, particularly during PCR amplification of the template fragments. If personalised genomics is to be realised we also need to ensure that protocols are standardised to minimise the run-to-run, and lab-to-lab variation that has been reported [Current “Next-generation” or “2reported .In the short to medium term, research groups with a finite budget have to make the decision whether to perform whole genome-sequencing on a small number of subjects, or exome-sequencing on a larger number, weighing up the relative merits of each along the way. Use of exome-seq has recently been supported by a report that the entire CCDS (a database of consensus coding sequences in human and mouse) exome can be interrogated with just 2.8 Gbp of sequence data, approximately 3% of the required data for whole genome shotgun experiments   that may well deliver on the promise to provide the $1000 genome. Single-molecule, real-time sequencing negates the need for amplification, removing one of the key sources of bias mentioned above. Companies such as Helicos and Pacific Biosciences [Second generation technology continues to evolve, with the main players continually improving both the hardware, and the chemistry that underpins sample preparation and the actually sequencing reaction itself. But already there are a number of technologies  phenotypes could help unravel the biological and functional sequence of DNA coding variants and might be translated to the clinical phenotype in the future. Whole genome sequencing is likely to become readily available at a reasonable cost and the genome/exome data could link genotype to the phenotype prospectively. The challenge would be to differentiate the associated alleles from the disease causing variants by means of genetic and biological studies.Despite the very large number of functional alleles identified by GWAS and the rapidly evolving field of functional genomics, their application in clinical cardiovascular medicine is limited at present. However, these approaches have led to an improvement of our understanding of CVD and can be translated to clinical utility. Gene-based pre-symptomatic prediction of illness, finer diagnostic sub-classifications and improved risk assessment tools will permit earlier and more targeted intervention. Pharmacogenetics will guide our therapeutic decisions and monitor response to therapy. Personalised medicine will require the integration of clinical information, stable and dynamic genomics, and molecular phenotyping.et al., disease-causing mutations for a wide range of Mendelian cardiac disorders have been revealed through linkage studies, thereby permitting screening of family members to identify mutation carriers for early intervention [These have already found a place in cardiovascular screening, diagnosis and pharmacogenesis. As recently reviewed by Kim rvention . A more rvention . P_0.001) [Non-invasive alternatives that reduce the need for invasive testing are a lucrative goal in clinical cardiology. Already in clinical use is the non-invasive diagnosis of cardiac allograft rejection with blood gene expression. The Invasive Monitoring Attenuation through Gene Expression: IMAGE trial compared the routine use of endomyocardial biopsies for monitoring rejection with a more selective use of endomyocardial biopsy guided by a gene-expression profiling test called AlloMap and noninvasive cardiac imaging. Both strategies resulted in equivalent clinical outcomes, but patients who were monitored with gene-expression profiling underwent far fewer biopsies per person-year of follow-up than did patients who were monitored with routine biopsy  .Pharmacogenomics is the study of how genetic variation affects the clinical response to drugs, with the implicit assumption that pharmacogenomic insights will enhance efficacy and reduce toxicity. It is likely to be the field that will benefit the most in the short term as it is easy to link genetics to the ability to metabolise a drug. Recognising that 25% of patients have a sub-therapeutic antiplatelet response to clopidogrel, researchers have identified several genetic variants affecting the metabolism of clopidogrel, a prodrug, to its active metabolite. Of these, the CYP2C19 variant allele has been best linked to impaired clopidogrel metabolism, reduced platelet inhibition, and a higher risk of adverse cardiovascular events after percutaneous coronary interventions . BecauseHence, clinical applications of genomics are already enhancing the practice of cardiovascular medicine. Advances in functional genomics will be increasingly important to practicing cardiovascular specialists in the coming years.Although the driving force behind GWASs was originally the discovery of novel biological pathways through the use of scalable genome technology and its capacity for generating hypothesis-independent genetic associations, the results have generated much excitement about the potential clinical applications of these genetic markers for disease prediction, prevention and diagnosis. It is hoped that genomic discoveries would lead to personalised medicine, whereby healthcare interventions would be guided based on individual’s genomic make up. Direct-to-consumer genetic testing companies now offer mutation analysis and SNP microarray sequencing based on the findings of traditional genetic research and GWASs before the clinical validity or utility of population screening is understood. The challenge will be to reconcile people’s concerns about genomic privacy and security with the need to allow researchers and clinicians data access. Other implications including life/health insurance, psychological risks to the patients if they know they have a genetic variant (that means a significant increase in risk) and family screening have to be considered as well. Conversely, knowing early in life of the predisposition to certain conditions, could lead to lifestyle modification, risk prevention and early intervention.Personalised medicine within cardiovascular medicine, has been emphasised in risk prediction models, especially coronary artery disease, where the goal is to identify individuals at risk so that early intervention and lifestyle modification can be initiated. Genotype based risk prediction is fixed from birth, allows early risk prediction, is less susceptible to biological variation over life and is easy to obtain with minimal measurement error. A series of recent studies attempted to demonstrate predictive utility of the 9p21 risk allele -96. All As we discern more genomic markers–SNPs, copy number variants, and rare alleles– that influence the development and course of cardiovascular disease, our goal as evidence-based clinicians will be to apply this knowledge judiciously and responsibly for more personalised and cost-effective care.Currently, there is an expanding gap between the availability of direct-to- consumer whole genome testing and physician knowledge regarding interpretation of test results. Advances in the genomic literacy of health care providers will be necessary for genomics to fulfil its potential to affect clinical practice.


Unlike the mammalian central nervous system (CNS), the CNS of echinoderms is capable of fast and efficient regeneration following injury and constitutes one of the most promising model systems that can provide important insights into evolution of the cellular and molecular events involved in neural repair in deuterostomes. So far, the cellular mechanisms of neural regeneration in echinoderm remained obscure. In this study we show that radial glial cells are the main source of new cells in the regenerating radial nerve cord in these animals.Holothuria glaberrima react to injury by dedifferentiation. Both glia and neurons undergo programmed cell death in the lesioned CNS, but it is the dedifferentiated glial subpopulation in the vicinity of the injury that accounts for the vast majority of cell divisions. Glial outgrowth leads to formation of a tubular scaffold at the growing tip, which is later populated by neural elements. Most importantly, radial glial cells themselves give rise to new neurons. At least some of the newly produced neurons survive for more than 4 months and express neuronal markers typical of the mature echinoderm CNS.We demonstrate that radial glial cells of the sea cucumber A hypothesis is formulated that CNS regeneration via activation of radial glial cells may represent a common capacity of the Deuterostomia, which is not invoked spontaneously in higher vertebrates, whose adult CNS does not retain radial glial cells. Potential implications for biomedical research aimed at finding the cure for human CNS injuries are discussed. The recent decades have seen an overturn of two major dogmas in neurobiology. First, it is now accepted that the central nervous system (CNS) can continue producing new cells after an animal reaches adulthood. Second, it is becoming increasingly clear that glial cells, besides performing auxiliary functions, have also other, more active, roles, one of which is involvement of glia in adult neurogenesis, either as stem cells per se or as “niche cells”, which regulate mitotic activity, fate decisions, and differentiation of the progenitor cells -3. ContiAlthough factors that suppress mammalian neural regeneration have been identified, and studies on fish and amphibians have significantly contributed to our understanding of the fundamental mechanisms of CNS repair, this body of experimental work has not yet translated into a meaningful therapeutic effect . In ordeAmong available non-chordate CNS regeneration models, echinoderms arguably represent the best match to the above two criteria. Modern molecular systematics places the phylum Echinodermata  as a sister group to chordates within the monophyletic taxon Deuterostomia -17 makinOur previous research ,26 has dHere, we provide a brief background on the organization of the sea cucumber central nervous system. For further details the reader is referred to Hyman , HeinzelAs has been noted elsewhere , althougIn our injury paradigm, one of the RNCs was cut at about the mid-body level Figure A, B and By this stage, the wide wound gap still persists at the site of the transection Figure A, but thBy days 8 – 12 after injury, the wound gap is still clearly discernible in the form of a deep furrow Figure A, B. TheAt this stage, the dedifferentiated tips of the radial nerve stumps form thin tubular outgrowths penetrating into the connective tissue that fills the wound gap Figure B-E’. It The ectoneural and hyponeural bands of the RNC form separate tubular rudiments that grow parallel to each other, however, the ectoneural band regenerates considerably faster Figure С.This is the stage when the two growing regenerates meet to bridge the injury gap and restore the anatomical continuity. The newly regenerated segment of the radial organs then starts to gradually resume its normal appearance Figure A – C. Th+ BrdU+ cells to the total number of BrdU+ cells, representing the contribution of glial cells to cell proliferation, remained essentially constant both in the uninjured and regenerating animals  Figure A with lae Figure C, D.+ progeny no longer showed positive staining with ERG1   assay to quantify the effect of induced programmed cell death on glial population of the RNC. These results are summarized in bar plots in Figure This paper presents data indicating that radial glial cells play a leading role during CNS regeneration in echinoderms. Echinoderm radial glial cells share a number of key characteristics with the radial glia of chordates. These include (a) an orthogonal orientation relative to the plane of the neuroepithelium; (b) elongated shape allowing the cell to stretch between the apical and basal surfaces of the neuroepithelium; (c) apical cilia protruding into the lumen of the epineural/hyponeural canal (in cryptosyringid echinoderms) or central canal (in chordates); (d) conspicuous bundles of intermediate filaments in the cytoplasm; (e) ability to produce and secrete Reissner’s substance ,21,22,24Synthesis of the data produced in the present study with our previous ultrastructural analysis of the regenerating CNS in sea cucumbers  suggestsIn regeneration-competent non-mammalian vertebrates, the cellular events, which are triggered in response to CNS injury, are similar to what we present here for echinoderms. Although some signs of reactive gliosis can be initially seen immediately after injury , this type of response eventually subsides, as the radial glia cells, which persist into adulthood in these animals, never form a glial scar. Instead, these cells seal the cut end of the stump and form glial outgrowths, which either grow caudally past the plane of amputation (in case of tail regeneration) or migrate into the lesion site and fill the injury gap . Most importantly, the activated glial cells are engaged in post-traumatic neurogenesis through extensive cell division and production of new functional neurons ,9-11,33.In mammals, CNS injury triggers a cascade of events, which constitute extensive reactive gliosis -36. RespThus, in different branches of the deuterostomian clade CNS injury elicits a response from glial cells surrounding the lesioned area. The analysis of the available data suggests that the nature of this initial glial response largely determines if scarring or complete regeneration will subsequently ensue. The permissive type of the glial response and production of new neurons is associated with the persistence of radial glial cells in the adult CNS both in echinoderms and regeneration-competent vertebrates. This implies that not only are the radial glial cells a common CNS component in different deuterostomes, but also that they play similar roles in post-traumatic regeneration in these taxa. It is interesting that after ~900 million years that have passed after separation of the clades Chordata and Amulacraria , the radOutside the deuterostomian lineage, glial cells do not necessarily constitute the cell source for nervous system regeneration and/or adult neurogenesis. Thus, cnidarians have an elaborate nervous system composed of well-developed nerve nets and nerve rings, but completely lack cells, which can be identified as glia ,7. In thThe very fact that the radial glial cells appear to be the only major cell source of CNS regeneration in echinoderms is even more interesting in view of well-known developmental plasticity of adult tissues in this phylum, where cells of different lineages can be recruited to rebuild lost or injured body parts. Thus, in some sea cucumbers, the mesothelium of the digestive tube  is able to transcend the germ layer boundaries and give rise to the luminal epithelium  in the anterior region of the regenerating gut, whereas in the posterior region the endodermal and mesodermal epithelia regenerated from their respective sources . By cont+, GABA+, Nurr1+, but not all neurons simultaneously. Third, the similarity of CNS regeneration at the cellular level calls for further comparative studies of molecular gene regulatory mechanisms underlying the plasticity of radial glia in echinoderms and regeneration-competent vertebrates. If many of the components of this molecular machinery are also evolutionary conserved and are present in poorly regenerating mammals, then there is certainly hope that it could be possible to convert the endogenous glial cells into neurons in the injured human central nervous system [The present study has exposed a number of issues that await further research. First, in mammals, the radial glia of the developing CNS and the astroglia of the adult nervous tissue are known to be heterogeneous in terms of their neurogenic potential and response to injury, respectively ,43. The s system  using reOur study demonstrates that echinoderms employ post-traumatic activation of radial glia to accomplish fast and efficient regeneration of their central nervous system, the process, which yields completely normal morphology and replaces the neurons lost to injury. Taken together, our data and the analysis of the available literature suggest that post-traumatic CNS regeneration via activation of radial glial cells is a conserved cellular mechanisms within the Deuterostomia, as it occurs both in echinoderms and regeneration-competent lower vertebrates. Since vestiges of this regenerative response can be seen even in poorly regenerating higher vertebrates, there is certainly hope that comparative studies can inform applied biomedical research how the phylogenetically conserved mechanisms, which might be present in a latent state, can be activated to improve neural repair in mammals, including human.Holothuria glaberrima Selenka, 1867 (Echinodermata: Holothuroidea) were collected at low tide from rocky shores adjacent to the La Perla and Piñones districts of northeast Puerto Rico and immediately brought to the laboratory. The surgery was performed as described previously [H. glaberrima poorly survives full-thickness injuries to the body wall, but readily regenerates as long as there is no direct communication between the coelom and the environment. The animals were then allowed to recover until they resumed the ability to attach themselves to the bottom or sides of the tanks and then transferred to larger tanks with fresh aerated seawater. During the first week after the surgery, the seawater was changed every day, and then weekly for the duration of the experiment.Adult individuals of eviously ,45. BrieFor general morphology, tissue samples of the uninjured and regenerating radial nerve cord were fixed in 4% paraformaldehyde with 0.1% glutaraldehyde and 0.2% picric acid in 0.01M PBS  overnight at 4°C. The samples were then rinsed in the same buffer, decalcified in 10% EDTA in 0.05 M Tris–HCl pH 7.4, dehydrated in alcohol, cleared in xylene, and embedded in paraplast (Sigma). Sections (10 μm) were collected on gelatin/chrome alum-coated slides, stained either with Mayer’s hematoxylin and eosin or with Giemsa stain (Sigma), and coverslipped in Permount (Fisher Scientific). Occasionally, cryosections (see below) were also stained with hematoxylin and eosin.For immunocytochemical analysis and cell counting assays (see below), uninjured and regenerating animals were sacrificed in groups of four. The tissue samples were fixed overnight with 4% paraformaldehyde in 0.01 M PBS at 4°C. After rinsing in the buffer, the samples were cryoprotected in graded sucrose solutions and frozen in OCT (Sakura Finetek). Cryosections (10 μm) were cut with a Leica CM1850 cryostat and collected on gelatinized slides. Immunostaining was performed as described elsewhere . BrieflyIn order to determine the percentage of dividing cells in the normal animals and at different time points of regeneration, we performed a single intracoelomic injection of 5-bromo-2-deoxyuridine . The BrdU-injected animals were returned to aquaria for 4 h and subsequently sacrificed. Tissues were processed for BrdU immunohistochemistry as described above.Alternatively, in order to trace the progeny of BrdU incorporating cells, the regenerating animals received BrdU injections (50 mg/kg) twice a day during days 8 thru 12 post-injury. This time frame corresponded to the period of active growth and the most extensive cell division in the regenerate (see the Results section). Tissue samples were fixed 12 hours, 51 days and 133 days after the last injection and then processed for BrdU immunohistochemistry as above.Deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was used to identify cells undergoing programmed cell death and was carried out using Fluorescein FragEL DNA Fragmentation Detection Kit  following the manufacturer’s manual.The sections were viewed and photographed with a Nikon Eclipse 600 microscope equipped with a Spot RT3 digital camera . Post-aquisition image processing included composite image (overlay) generation, assembling panoramic images, brightness/contrast adjustment, multi-panel figure assembling and annotation and was carried out using the open-source software tools Fiji  and GIMPCells were counted on every third serial longitudinal section using micrographs taken with a 100× objective  in the normal and regenerating RNC.Click here for fileANOVA test results for dynamics of cell proliferation.Click here for fileQuantification of programmed cell death (through TUNEL assay) in the normal and regenerating radial nerve cord.Click here for fileANOVA test results for dynamics of programmed cell death.Click here for fileRepresentative example of a micrograph used in cell counting assays. The micrograph was taken with a 100x objective. Only cells in sharp focus were counted (such as those indicated by arrows). A shows co-localization of ERG1 and BrdU labeling in the cells marked with arrows, whereas A’ and A” show these two type of labeling in separate channels.Click here for file


Major advances in various disciplines of basic sciences including embryology, molecular and cell biology, genetics, and nanotechnology, as well as stem cell biology have opened new horizons for regenerative therapy. The unique characteristics of stem cells prompt a sound understanding for their use in modern regenerative therapies. This review article discusses stem cells, developmental stages of the eye field, eye field transcriptional factors, and endogenous and exogenous sources of stem cells. Recent studies and challenges in the application of stem cells for retinal pigment epithelial degeneration models will be summarized followed by obstacles facing regenerative therapy. Regenerative medicine intends to provide therapies for severe injuries or chronic diseases in which endogenous repair does not sufficiently restore the damaged tissue. These include congestive heart failure, osteoporosis, spinal cord injuries, Alzheimer’s and Parkinson‘s diseases, age-related macular degeneration, and retinitis pigmentosa.1Age-related macular degeneration (AMD) affects 10% to 20% of individuals over 65 years of age and is the leading cause of severe visual impairment in the elderly in industrialized nations. There are many senescent changes in the normal pigment epithelium including a decrease in retinal pigment epithelium (RPE) density, a clinically observed decrease in the pigmented appearance of RPE cells, and the accumulation of lipofuscin within RPE cells.Hereditary retinal degenerations are one of the major causes of blindness. This group of disorders includes retinitis pigmentosa (RP) which occurs at an incidence of 1 in 2000 individuals.Approaches to regeneration which have been pursued include the following:Promotion of endogenous regeneration via use of growth factors.Exogenous delivery of living cells of allogenic and/or autologous origin, particularly stem cells because of their plasticity and capacity for self-renewal.Integration of the above concepts to reach the goal of successful transplantation and development of cells.1Stem cells have attracted considerable attention, not only as a means of understanding metazoan development, but also as potential therapeutic agents for a spectrum of currently untreatable diseases.A number of transcription factors begin to express early in the embryonic phase of development, these factors mark the region which is going to constitute the future eyes and are called the eye field transcription factors (EFTF). The EFTFs that are expressed early in eye field include Rx, Pax6, Six3, Lhx2, and Optx2 (Six6).8The eye field forms late in gastrulation, it takes shape at the anterior end of the neural plate in the diencephalic region of the forebrain. The eye field initially extends across the midline as a single domain. This single field is subsequently split into two lateral domains due to the repression of EFTFs by the Sonic hedgehog homolog (SHH), which is released from the prechordal mesoderm at the midline.9Sonic hedgehog is an extracellular glycoprotein important in several other inductive events throughout the embryonic phase. The EFTFs are essential for eye development; mutations in each of these genes are associated with either anophthalmia or microphthalmia.10Prior to development of the eye field, the anterior nervous system becomes distinct from the posterior. The Otx2 transcription factor (a member of the orthodenticle family) is critical in the control of this distinction.12Rx gene in mice result in anophthalmia, with no eye development after the optic vesicle stage.Xenopus embryos using morpholino oligonucleotides against the Xenopus homolog to Rx.15Homozygous null mutations of the Rx in Xenopus embryos results in hyperproliferation of the neural retina and RPE, as well as formation of ectopic retinal tissue.Rx gene in humans has also been identified in a patient suffering from anophthalmia and sclerocornea.16Overexpression of Pax6 is the master regulatory gene in eye development. It belongs to the family of paired box homeodomain genes and has been highly conserved across species. Pax6 is expressed in the anterior neural plate at the end of gastrulation and then becomes restricted to the region of the optic vesicle as well as the lens ectoderm. Its expression persists throughout optic development and ultimately into adult animals in ganglion, horizontal, and amacrine cells.18The most well studied EFTF is Pax6. It has been proposed that Pax6 result in a variety of phenotypes, depending on gene dosage. Homozygous mutations causing a complete loss of Pax6 expression result in anophthalmia in mice and rats.Pax6 mutant mouse embryos have normal Rx expression, suggesting that Pax6 is downstream of Rx.DrosophilaXenopusPax6 in Xenopus results in multiple ectopic eyes along the dorsal central nervous system (CNS) along with ectopic expression of other EFTFs including Rx in these areas. This finding suggests that Pax6 plays a role in the induction of Rx.Pax6 is a master regulatory gene during eye development.Mutations in Lhx2 is an EFTF that belongs to the family of Lim-homeodomain genes. It is expressed in the optic vesicles just before the completion of gastrulation.23Lhx2 may lie downstream of Pax6. Overexpression of Lhx2 in Xenopus embryos results in large eyes as well as ectopic retinal tissue.24Lhx2 null mutants fail to form eyes.Six3 belongs to the Six-homeodomain family of genes. Six3 appears in the region of the presumptive eye field around the same time as Pax6.27Six3 inactivation in Medaka fish has been shown to result in anophthalmia and forebrain agenesis.Six3 in Medaka fish results in multiple eye-like structures that express other EFTFs30Optx2  also belongs to the Six-homeodomain family of genes and is expressed from the optic vesicle stage.Xenopus embryos result in large expansion of the retinal domain as well as hyperproliferation of cultured retinal progenitors transfected with XOptx2.36Wnt signalling has a key role in early embryonic patterning through the regulation of cell fate decisions, tissue polarity, and cell movements. In the nervous system, Wnt signalling also regulates neuronal connectivity by controlling axonal path finding and remodelling, dendrite morphogenesis, and synapse formation .Rx and Six3 expression when overexpressed in Xenopus embryos. On the other hand, Wnt-11, which works through the non-canonical pathway, results in larger eyes in Xenopus when overexpressed.38Although a considerable amount has been learned about the role of EFTFs in ocular development, little is known about the factors that control their expression. Recently, a few investigators have looked into the role of Wnt signaling in the initiation and regulation of the eye fields.Xenopus results in formation of multiple ectopic eyes. Fz-3 is believed to preferentially activate the non-canonical Wnt pathway.Xenopus eye formationRx expression in Xenopus. Loss of EAF2 function results in loss of the eyes, while loss of Wnt-4 function can be rescued by EAF2 misexpression in frogs. Cell–cell signaling is also critical for movement of eye field precursors to the correct locations prior to activation of EFTFs. In Xenopus, all cells destined to form the eye field accumulate together through ephrin-B1 signaling. This can be inhibited by fibroblast growth factors (FGFs); activated FGF receptors modulate the activity of ephrin-B by phosphorylating their intracellular domain.Misexpression of the Frizzled-3 (Fz-3) Wnt receptor in Wnt-3a, in combination with bone morphogenetic proteins and Sonic hedgehog, induces differentiation of ESCs into interneurons. These studies raise the exciting possibility that manipulation of Wnt signaling could provide the means for stem cell expansion or differentiation. Low molecular weight inhibitors of glycogen synthase kinase-3β mimic these effects and similarly promote the proliferation of Muller glia-derived progenitors.Neuronal progenitors with retinal potential as adult stem cell types reside in:Ciliary body epithelium.42Iris pigment epithelium.43The peripheral margin of the postnatal retina.44For many decades, it was believed that neurons in the adult mammalian central nervous system could not regenerate after injury, as postulated by Ramon y CajalIn vivo labeling analysis with bromodeoxyuridine (BrdU) shows that the pigmented portion of the ciliary body in adult rats contains cells with proliferative potential.in vitro by reprogramming or de-differentiation. Similar mechanisms have been invoked to explain the conversion of oligodendrocytic progenitors into neural stem cells.The question of whether adult derivatives of mammalian retinal neuroepithelium harbor cells with stem cell properties was recently addressed. Two laboratories showed that in the adult mammalian eye, the ciliary epithelium but not the neural retina, contains neural progenitors.Muller glial cells have also been demonstrated to possess retinal progenitor properties in the adult retina.56Studies indicate that Muller glia proliferate and de-differentiate into retinal progenitors in response to retinal damage and then migrate and differentiate into retinal neurons. After proliferation in response to retinal damage, BrdU-labeled cells migrate into the retinal neuron-specific layer and differentiate into cells positive for retinal neuron-specific markers such as rhodopsin (rod photoreceptors) and PKCa (rod bipolar cells). The Muller glia-derived progenitors appear to differentiate into the type of cell that was damaged, evidenced by the fact that Muller glia mainly generate photoreceptors in the photoreceptor–damaged retina. Exogenous application of retinoic acid and Sonic hedgehog induces photoreceptor differentiation from Muller glia-derived progenitors, similar to that observed with retinal progenitors during eye development.57In retinal regeneration, the differentiation of Muller glia-derived progenitors can be regulated by both intrinsic and extrinsic factors, similar to what has been observed with retinal progenitors during eye development.58Growth factors and neurotrophins such as FGF263In vitro studies in chick retinae suggest that CNTF may play a transient role in photoreceptor development by increasing the number of opsin-expressing cells.67Rod photoreceptor development is promoted by bFGF (FGF2), Sonic hedgehog, taurine, and laminin beta 2.A phase 1 trial with GDNF and CNTF in patients with retinitis pigmentosa, indicated that CNTF is safe for the human retina, even with severely compromised photoreceptors, and may promote visual improvement.68Stem cells are of interest because of their plasticity and capacity to self-renew, as well as to give rise to specialized cell types. Stem cells remain uncommitted and self-renewable until they receive a signal to develop into distinct cell types.70ESCs and EGCs appear very similar and are likely to have comparable medical applications. In fact, a recent report indicates that ESCs, which are derived from the inner cell mass of early embryos, most closely resemble EGCs.ESCs apparently can self-renew in cultures without limit, although the mechanisms underlying this capacity are not yet fully understood.78In the following section, we will review recent studies that concern challenges surrounding exogenous delivery and transplantation of stem cells.in vivo can rescue vision in dystrophic Royal College of Surgeons (RCS) rats. Fifteen human embryonic stem cell (hESC) lines were derived from human frozen blastocysts or cleaved embryos that were donated by couples who had completed their fertility treatment. Human ESCs were cultured and allowed to spontaneously differentiate; this resulted in appearance of RPE clusters over the course of 6 to 8 weeks, from which hESC-derived RPE cells were isolated and subcultured. Dystrophic RCS rats which had undergone immune suppression, received injections of hESC-derived RPE cells into the subretinal space, between the RPE and photoreceptor layers on postnatal day 21, at an age when photoreceptor degeneration had yet to develop. Electroretinography (ERG) responses were tested on postnatal days 60 and 90. By 60 days, the hESC-derived RPE grafted animals achieved significantly better ERG responses than untreated and sham-injected animals. For the optomotor test, which provided a measure of spatial acuity, in sham treated rats, a threshold response of 0.29±0.03 cycle/degrees (c/d) was recorded on day 100; untreated animals revealed a figure of 0.21±0.03 c/d. By contrast, the cell-grafted rats demonstrated levels of 0.42±0.03 c/d, significantly better than sham injected rats. Anatomical examination showed photoreceptor rescue, 5 to 7 cells deep in the outer nuclear layer (ONL). The ONL was 10 to 12 cells in nondystrophic rats, while in dystrophic rats this layer was reduced to one cell deep on day 100. Improvement in visual performance was 100% better than untreated controls  without evidence of untoward pathology.Lund and colleaguesIn another studyThe same authorsLimited clinical trials of retinal implantation have been underway since 1998. Between January 1, 1998 and January 1, 2001, a phase 1 clinical trialSignificant challenges of replacement therapy include the following:Efficient tissue delivery.Immune surveillance.Maintenance of an appropriate state of differentiation by transplanted tissue.Integration of the transplant with the host and reestablishment of functional synaptic circuitry.As mentioned before, there are a handful of sources for immortalized cell lines for efficient tissue delivery. In the case of RPE cell replacement therapy, these include ARPE-1992OCT-3/4, SOX2, and KLF4. Treating iPSCs with Wnt and Nodal antagonists in a suspension culture induced the expression of retinal progenitor cell markers and generated RPE cells. Subsequently, treatment with retinoic acid and taurine, generated cells positive for photoreceptor markers in all but one human cell lines. The authors concluded that the efficiency of differentiation into RPE and photoreceptors from human iPSC cultures of cell lines 201B7 and 253G1 was not different from that of human ESC cultures. Therefore, in addition to their similarity in pluripotency, retinal differentiation methods using specific factors for ESCs are also applicable to iPSCs, although further research will be required to determine the function of induced retinal cells. As for immune rejection, transplantation of patient-specific cells will have several advantages over comparable differentiated ESCs and allogenic RPE transplantation, which may induce immune rejection of the graft tissue in the absence of systemic immune suppression.Hirami et alin vivo, Carr et alin vitro and in vivo aspects. They examined the potential of human iPSCs to differentiate into fully characterized RPE cells (iPSC-RPE). Furthermore they analyzed their functionality in vitro and in vivo after transplantation of iPSC-RPE cells into dystrophic RCS rat. The iPSCs derived from IMR-90 human fetal lung fibroblast cell line2 tissue culture flask with no evidence of cell multi-layering. The appearance of dome-shaped blisters suggested that fluid transport from apical to basal cell surfaces might be occurring. The appearance of RPE cell morphology was associated with the expression of a panel of classic RPE genes and proteins required for retinoid recycling , phagocytosis (FAK and MERTK), and melanogenesis . Cells also expressed the anti-neovascular agent/neurotrophic factors pigment epithelium-derived factor (PEDF), and Krt8, an epithelial keratin associated with RPE cell proliferation.SOX2 and NANOG expression, indicative of differentiation away from the iPSC phenotype. Pax6, Otx2, and MITF, transcription factors involved in RPE cell development, were localized in the nucleus, whilst Rlbp1, Pmel17 and bestrophin-1 were expressed cytoplasmically. Importantly, differentiated cells were negative for RPE cell de-differentiation markers Krt8 and Ki-67, markers of the active phase of the cell cycle, indicating that cells were no longer proliferative. They used phagocytosis assays to assess the functional potential of cells. The iPSC-RPE cells were able to phagocytose fluorescent labeled porcine photoreceptor outer segments (POS) in co-culture. The apical surfaces of the cells envelop the POS and internalized coated pits are seen after 3 hours co-culture, with end-stage lipid deposits observed after 12 hours. For in vivo assessment, after 20 hours of transplantation into the subretinal space of the eyes of RCS rats, they observed a layer of pigmented cells within the subretinal space, which were Ki-67 negative. The origin of the cells was confirmed by staining with human-specific markers (HSM). At 8 days, iPSC-RPE was no longer present as a layer, but formed cell boluses. However, transplanted cells maintained expression of RPE markers such as Rlbp1 and Otx2, with only occasional cells positive for Ki-67. The in vivo expression of RPE65 was not detectable by immunocytochemistry. The pattern of staining displayed by transplanted iPSC-RPE cells in vivo was similar to that reported recently for hESC-RPE grafted into dystrophic RCS rat retinae.in vivo, cells at the outside edge of the iPSC-RPE cell bolus could phagocytose photoreceptor outer segments from the RCS rat, as indicated by the presence of rhodopsin-positive photoreceptor material within the cellular membrane of iPSC-RPE cells labeled with HSM. To assess visual function, they used the head-tracking response of RCS dystrophic rats 13 weeks after receiving subretinal iPSC-RPE transplantation in one eye only. Preservation of higher spatial frequency (0.5 c/d) monocular optokinetic head-tracking response was associated with the iPSC-RPE transplanted eye when compared with sham injected eyes and non-transplanted eyes. Even though the ONL was preserved 13 weeks post-transplantation, there was little evidence of surviving iPSC-RPE cells within the subretinal space by this time. Occasional HSM-positive material could be detected in the subretinal space, but the lack of a defined membrane and the absence of DAPI staining suggest that these cells were not viable. However, the investigators did find host cells positive for the monocyte/ macrophage marker CD68 within the subretinal graft site, which did not stain for HSM. At 8 days, these cells had a clear appearance, and rhodopsin-positive material was found within the CD68 expressing cells. The absence of iPSC-RPE cells in the subretinal space at the time of functional assessment (13 weeks) indicates that the significant benefits observed could not be wholly attributed to donor cells. Although all animals were maintained on oral cyclosporin throughout the experiment, this was not sufficient to sustain iPSC-RPE cell survival. These findings are in agreement with previous studies that show that xenografts can be compromisedTo show the protective effects of human iPSC-derived RPE cell transplantation While this particular line of iPSC-RPE cells could not be used as direct therapy due to viral insertions of pluripotency genes, recent advances in iPSC reprogramming technology, including the use of small moleculesAt last, the most challenging and yet unresolved part, is how to integrate the transplanted tissue within the host in the most effective manner and reestablish the functional synaptic circuitry within the tissue.This review presents data on what has been achieved in the field of regenerative therapy in retinal diseases, a summary of the developmental literature, ongoing research on development of therapeutically useful ocular cell types (RPE cells and photoreceptors) and obstacles in the use of stem cells.One of the most prominent achievements is the successful differentiation of iPSCs into RPE cells. The major benefit of using iPSCs to treat AMD is that a patient-specific therapy may help eliminate problems associated with immune rejection. The proof of concept for the therapeutic use of a patient’s own iPSC-derived RPE lies in current clinical treatments for AMD. Although, iPSC-RPE may carry the same genetic defect responsible for AMD in the patient, the fact that these cells have not been diseased by age, like macular RPE, suggests that they could still be used as a viable therapeutic modality. iPSC therapy might also be useful in patients with genetic diseases, such as Leber’s congenital amaurosis where transplantation could be combined with gene therapy to correct the inherent genetic defects.96A major issue facing stem cell research, and retinal transplantation of stem cells in particular, is the question of functional integration of grafted cells. This issue incorporates the question of phenotypic maturation of stem cells, particularly into functional neurons and photoreceptors, as well as the question of how surviving host circuits react to the presence of newly integrated donor cells. For instance, the possibility has frequently been expressed that such cells may integrate incorrectly. On the other hand, donor cells might successfully create new functional circuits, improve host signaling, or induce rescue effects. These possibilities should all be investigated in multiple settings since results tend to vary considerably between models.6While much remains to be demonstrated, particularly in terms of potential visual benefits, retinal stem cell transplantation provides an exciting new strategy for treatment of degenerative retinal disease and offers hope that effective treatment may be within reach.