Abstract: The translation of current tissue engineering approaches to clinical application is somehow limited by the use of scaffolding materials. Recently a number of in vitro scaffold-free three-dimensional culture techniques have been developed. These techniques realize the assembly of tissue-like structures including but not limited to spheroids, blood vessels and cartilage. In particular, cells can now self-assemble to form planar tissue-like structures at the interface of an aqueous-two-phase system (ATPS). The unique advantage of this technique is that without a solid substrate, planar tissue-like structures can now be assembled rapidly with very simple procedures. This technique can potentially be very useful for tissue engineering in eye because of its ability to direct cells to form monolayer. In this talk, we will introduce what ATPS is and its current applications in biomedical research. We will then present an approach to assemble cell sheets in ATPS using both primary cells isolated from porcine eyes and other cell lines. The physiological relevance of these eye-related cell sheets as well as their potentials in ophthalmic research and applications will be discussed.

Abstract: The translation of current tissue engineering approaches to clinical application is somehow limited by the use of scaffolding materials. Recently a number of in vitro scaffold-free three-dimensional culture techniques have been developed. These techniques realize the assembly of tissue-like structures including but not limited to spheroids, blood vessels and cartilage. In particular, cells can now self-assemble to form planar tissue-like structures at the interface of an aqueous-two-phase system (ATPS). The unique advantage of this technique is that without a solid substrate, planar tissue-like structures can now be assembled rapidly with very simple procedures. This technique can potentially be very useful for tissue engineering in eye because of its ability to direct cells to form monolayer. In this talk, we will introduce what ATPS is and its current applications in biomedical research. We will then present an approach to assemble cell sheets in ATPS using both primary cells isolated from porcine eyes and other cell lines. The physiological relevance of these eye-related cell sheets as well as their potentials in ophthalmic research and applications will be discussed.

Abstract: Encapsulated-cell therapy (ECT) is an attractive approach for continuously delivering freshly synthesized therapeutics to treat sight-threatening posterior eye diseases, circumventing repeated invasive intravitreal injections and improving local drug availability clinically. Composite collagen-alginate (CAC) scaffold in ECT contains an interpenetrating network that integrates the physical and biological merits of its constituents, including biocompatibility, mild gelling properties and availability. An injectable CAC system that supported the growth of HEK293 cells with sustainable glial-derived neurotrophic factor (GDNF) delivery has been developed. Continuous GDNF delivery was detected in culture and in healthy rat eyes for at least 14 days. The gels were well tolerated with no host tissue attachment and contained living cell colonies. Most importantly, gel implantation in dystrophic Royal College of Surgeons rat eyes for 28 days retained photoreceptors while those gels containing higher initial cell number yielded better photoreceptor rescue effect. This rescue effect is clinically relevant as photoreceptor death is a common pathology in many retinal diseases. Moreover, since cells including autologous cells can be genetically engineered to secrete various therapeutic agents, CAC gel offers a flexible system design and is a potential treatment option for other chronic neurodegenerative diseases.

Abstract: Encapsulated-cell therapy (ECT) is an attractive approach for continuously delivering freshly synthesized therapeutics to treat sight-threatening posterior eye diseases, circumventing repeated invasive intravitreal injections and improving local drug availability clinically. Composite collagen-alginate (CAC) scaffold in ECT contains an interpenetrating network that integrates the physical and biological merits of its constituents, including biocompatibility, mild gelling properties and availability. An injectable CAC system that supported the growth of HEK293 cells with sustainable glial-derived neurotrophic factor (GDNF) delivery has been developed. Continuous GDNF delivery was detected in culture and in healthy rat eyes for at least 14 days. The gels were well tolerated with no host tissue attachment and contained living cell colonies. Most importantly, gel implantation in dystrophic Royal College of Surgeons rat eyes for 28 days retained photoreceptors while those gels containing higher initial cell number yielded better photoreceptor rescue effect. This rescue effect is clinically relevant as photoreceptor death is a common pathology in many retinal diseases. Moreover, since cells including autologous cells can be genetically engineered to secrete various therapeutic agents, CAC gel offers a flexible system design and is a potential treatment option for other chronic neurodegenerative diseases.

Abstract: Animals promote their survival by avoiding rapidly approaching objects that indicate threats. It is believed that looming cues are detected by retinal ganglion cells (RGCs) that project to the superior colliculus (SC). However, the exact type of RGC that transmits looming-related signals remains unclear. Here we identify a specific transient type of RGCs that controls mouse looming-evoked defensive response by sending axonal collaterals to the dorsal raphe nucleus (DRN) and SC. Looming signals transmitted by DRN-projecting RGCs activate DRN GABA neurons and in turn inhibit serotonin neurons. Moreover, optogenetically stimulating serotonin neurons reduces looming-evoked defensive behaviors. Thus, a dedicated population of RGCs detects rapidly approaching visual threats and their input to the DRN controls a serotonergic self-gating mechanism that regulates innate defensive responses. Our study provides new insights into how DRN and SC work in concert to extract and translate visual threats into defensive behavioral responses.

Abstract: Animals promote their survival by avoiding rapidly approaching objects that indicate threats. It is believed that looming cues are detected by retinal ganglion cells (RGCs) that project to the superior colliculus (SC). However, the exact type of RGC that transmits looming-related signals remains unclear. Here we identify a specific transient type of RGCs that controls mouse looming-evoked defensive response by sending axonal collaterals to the dorsal raphe nucleus (DRN) and SC. Looming signals transmitted by DRN-projecting RGCs activate DRN GABA neurons and in turn inhibit serotonin neurons. Moreover, optogenetically stimulating serotonin neurons reduces looming-evoked defensive behaviors. Thus, a dedicated population of RGCs detects rapidly approaching visual threats and their input to the DRN controls a serotonergic self-gating mechanism that regulates innate defensive responses. Our study provides new insights into how DRN and SC work in concert to extract and translate visual threats into defensive behavioral responses.

Abstract: Vision loss in retinal disease is often secondary to neural cell loss. Neural loss of any type including that of the retina has always been considered irreversible as these cells rarely retain the ability to regenerate. The recent identification of stable stem cell sources and the advances in stem cell technology have transformed this area of research science into an important area of strong therapeutic possibility. These sources include human embryonic stem cells (hESC), induced pleuripotent stem cell sources (iPS) as well as adult sources. The main advantage of using a stem cell source is that there is an infinite capacity to reproduce and therefore an infinite capacity to produce cells, including neural cells for transplantation. The challenge more recently has been to transform these stem cells into differentiated cells that are useful for transplantation in disease. In terms of the retina, hESC have been successfully developed into retinal pigment epithelial cells. These cells have been characterised as identical to native human RPE cells structurally, functionally and biochemically. Previous studies of macular translocation and RPE/choroidal transplantation have shown that vision loss from AMD can be reversed. Early animal studies show that the transplanted HESC RPE survive and can prevent vision loss in animal models of disease. Initial hESC based RPE transplantation trials using suspension cultures were successful in demonstrating safety of the cells in the context of disease and sub-retinal delivery. More recently, we have carried out the first 2 transplantations of sheets of hESC based RPE on a coated artificial Bruch’s membrane, in the London Project’s RPE transplantation trial, with promising results. As well as RPE— Bruch’s transplantation I will also briefly discuss the recent advances in neuro-retinal and vascular reconstructions using stem cells.

Abstract: Vision loss in retinal disease is often secondary to neural cell loss. Neural loss of any type including that of the retina has always been considered irreversible as these cells rarely retain the ability to regenerate. The recent identification of stable stem cell sources and the advances in stem cell technology have transformed this area of research science into an important area of strong therapeutic possibility. These sources include human embryonic stem cells (hESC), induced pleuripotent stem cell sources (iPS) as well as adult sources. The main advantage of using a stem cell source is that there is an infinite capacity to reproduce and therefore an infinite capacity to produce cells, including neural cells for transplantation. The challenge more recently has been to transform these stem cells into differentiated cells that are useful for transplantation in disease. In terms of the retina, hESC have been successfully developed into retinal pigment epithelial cells. These cells have been characterised as identical to native human RPE cells structurally, functionally and biochemically. Previous studies of macular translocation and RPE/choroidal transplantation have shown that vision loss from AMD can be reversed. Early animal studies show that the transplanted HESC RPE survive and can prevent vision loss in animal models of disease. Initial hESC based RPE transplantation trials using suspension cultures were successful in demonstrating safety of the cells in the context of disease and sub-retinal delivery. More recently, we have carried out the first 2 transplantations of sheets of hESC based RPE on a coated artificial Bruch’s membrane, in the London Project’s RPE transplantation trial, with promising results. As well as RPE— Bruch’s transplantation I will also briefly discuss the recent advances in neuro-retinal and vascular reconstructions using stem cells.

Abstract: Myopia and astigmatism, two common refractive errors frequently co-exist, are degrading vision at all working distances in populations worldwide. Eyeballs having high degrees of myopia and astigmatism are known to exhibit abnormal eye shape at the anterior and posterior eye segments, but whether the outer coats of these abnormal eyeballs, cornea anteriorly and sclera posteriorly, are regulated by region-specific molecular mechanism remains unclear. Here we presented the changes in eye shape and mRNA expression levels of three genes (MMP2, TIMP2, and TGFB2), all known to participate in extracellular matrix organization, at five regions of the cornea and sclera in chickens developing high myopia and astigmatism induced by form deprivation. Our results showed that, compared to normal chicks, the highly myopic-astigmatic chicks had significantly astigmatic cornea, deeper anterior chamber, longer axial length, and higher expressions of all three genes in the superior sclera. These results imply that local molecular mechanism may manipulate the eye’s structural remodeling across the globe during refractive eye growth.

Abstract: Myopia and astigmatism, two common refractive errors frequently co-exist, are degrading vision at all working distances in populations worldwide. Eyeballs having high degrees of myopia and astigmatism are known to exhibit abnormal eye shape at the anterior and posterior eye segments, but whether the outer coats of these abnormal eyeballs, cornea anteriorly and sclera posteriorly, are regulated by region-specific molecular mechanism remains unclear. Here we presented the changes in eye shape and mRNA expression levels of three genes (MMP2, TIMP2, and TGFB2), all known to participate in extracellular matrix organization, at five regions of the cornea and sclera in chickens developing high myopia and astigmatism induced by form deprivation. Our results showed that, compared to normal chicks, the highly myopic-astigmatic chicks had significantly astigmatic cornea, deeper anterior chamber, longer axial length, and higher expressions of all three genes in the superior sclera. These results imply that local molecular mechanism may manipulate the eye’s structural remodeling across the globe during refractive eye growth.

Abstract: Myopia prevalence is dramatically increasing in recent years and in cases in which the refractive error is greater than ?6.00 D this disease can lead to severe visual impairment as well as even blindness. Changes in visual input affect the balance between ocular growth and refractive power development. If a mismatch occurs during eye development, the severity of this error affects the degree of myopia. In different animal models of this disease, we found that spatial visual stimuli are essential for maintaining a stable refractive status and normal vision. This is evident because the effects of changes in temporal visual stimuli (e.g., flickering light) on this process depend on whether spatial information is present or absent in the visual environment. Furthermore, the frequency, wavelength and intensity of light are involved in controlling refraction development. However, the molecular mechanisms underlying light-induced refraction changes are still unclear. There is definitive evidence that dopamine (DA) is one of the regulators of this process. This retinal neurotransmitter released by dopaminergic amacrine cells appears to play an important role in vision-guided eye growth because its synthesis and release are positively associated with the light intensity and spatial stimuli impinging on the retina. We found that bright light enhances retinal DA synthesis, and attenuates form deprivation myopia (FDM) development via activation of the dopamine receptor 1 (D1R). A nonselective DA receptor agonist apomorphine (APO) inhibited FDM in dopamine receptor 2 (D2R) knockout mice. These individual similar effects of DA and APO in wildtype and D2R knockout mice suggest that D1R activation has a protective effect against myopia development. On the other hand, D2R activation instead appears to promote myopia development because either genetic D2R ablation or pharmacological inactivation of D2R also attenuates myopia development. Based on these results, we hypothesize that the visual environment regulates the retinal DA levels, which in turn affects the relative balance between D1R and D2R activation. When D1R is relatively hyperactivated, the ocular refractive status shifts towards hyperopia. In contrast, such an effect on D2Rpromotes the refractive status to shift in the opposite direction towards myopia.

Abstract: Myopia prevalence is dramatically increasing in recent years and in cases in which the refractive error is greater than ?6.00 D this disease can lead to severe visual impairment as well as even blindness. Changes in visual input affect the balance between ocular growth and refractive power development. If a mismatch occurs during eye development, the severity of this error affects the degree of myopia. In different animal models of this disease, we found that spatial visual stimuli are essential for maintaining a stable refractive status and normal vision. This is evident because the effects of changes in temporal visual stimuli (e.g., flickering light) on this process depend on whether spatial information is present or absent in the visual environment. Furthermore, the frequency, wavelength and intensity of light are involved in controlling refraction development. However, the molecular mechanisms underlying light-induced refraction changes are still unclear. There is definitive evidence that dopamine (DA) is one of the regulators of this process. This retinal neurotransmitter released by dopaminergic amacrine cells appears to play an important role in vision-guided eye growth because its synthesis and release are positively associated with the light intensity and spatial stimuli impinging on the retina. We found that bright light enhances retinal DA synthesis, and attenuates form deprivation myopia (FDM) development via activation of the dopamine receptor 1 (D1R). A nonselective DA receptor agonist apomorphine (APO) inhibited FDM in dopamine receptor 2 (D2R) knockout mice. These individual similar effects of DA and APO in wildtype and D2R knockout mice suggest that D1R activation has a protective effect against myopia development. On the other hand, D2R activation instead appears to promote myopia development because either genetic D2R ablation or pharmacological inactivation of D2R also attenuates myopia development. Based on these results, we hypothesize that the visual environment regulates the retinal DA levels, which in turn affects the relative balance between D1R and D2R activation. When D1R is relatively hyperactivated, the ocular refractive status shifts towards hyperopia. In contrast, such an effect on D2Rpromotes the refractive status to shift in the opposite direction towards myopia.

Abstract: Dopamine is known as a key molecule in retinal signaling pathways regulating visually guided eye growth, as evidenced by reduced retinal dopamine levels in various species when experimental myopia is generated. However, in C57BL/6 mice our recent work demonstrated that neither retinal dopamine levels, retinal tyrosine hydroxylase (rate-limiting enzyme in dopamine synthesis) levels, nor dopaminergic amacrine cell density/morphology, were altered during the development of form-deprivation myopia (FDM). These results suggest that retinal dopamine is unlikely associated with FDM development in this mouse strain. The role of dopamine in refractive development was further explored in this mouse strain when retinal dopamine levels were reduced by intravitreal injections of 6-OHDA, a neurotoxin that specifically destroys dopaminergic neurons. The dose was so chosen that retinal dopamine levels were reduced, but no significant changes in electroretinographic responses were detected. 6-OHDA induced significant myopic shifts in refraction in a dose-dependent manner, suggesting the involvement of dopamine in normal refractive development. Biometric measurements of ocular dimensions revealed that 6-OHDA resulted in a shorter axial length and a steeper cornea, while form-deprivation led to a longer axial length without changing the corneal radius of curvature. These results strongly suggest that in addition to the dopamine-independent mechanism, a dopamine-dependent mechanism works for refractive development. We have obtained evidence, suggesting that the dopamine-independent mechanism might be related to intrinsically photosensitive retinal ganglion cells (ipRGCs). Firstly, selective ablation of ipRGCs with an immunotoxin resulted in myopic shifts in refraction. Secondly, form-deprivation induced less myopic shifts in animals with ipRGC ablation.

Abstract: Dopamine is known as a key molecule in retinal signaling pathways regulating visually guided eye growth, as evidenced by reduced retinal dopamine levels in various species when experimental myopia is generated. However, in C57BL/6 mice our recent work demonstrated that neither retinal dopamine levels, retinal tyrosine hydroxylase (rate-limiting enzyme in dopamine synthesis) levels, nor dopaminergic amacrine cell density/morphology, were altered during the development of form-deprivation myopia (FDM). These results suggest that retinal dopamine is unlikely associated with FDM development in this mouse strain. The role of dopamine in refractive development was further explored in this mouse strain when retinal dopamine levels were reduced by intravitreal injections of 6-OHDA, a neurotoxin that specifically destroys dopaminergic neurons. The dose was so chosen that retinal dopamine levels were reduced, but no significant changes in electroretinographic responses were detected. 6-OHDA induced significant myopic shifts in refraction in a dose-dependent manner, suggesting the involvement of dopamine in normal refractive development. Biometric measurements of ocular dimensions revealed that 6-OHDA resulted in a shorter axial length and a steeper cornea, while form-deprivation led to a longer axial length without changing the corneal radius of curvature. These results strongly suggest that in addition to the dopamine-independent mechanism, a dopamine-dependent mechanism works for refractive development. We have obtained evidence, suggesting that the dopamine-independent mechanism might be related to intrinsically photosensitive retinal ganglion cells (ipRGCs). Firstly, selective ablation of ipRGCs with an immunotoxin resulted in myopic shifts in refraction. Secondly, form-deprivation induced less myopic shifts in animals with ipRGC ablation.

Abstract: Axon regeneration capacity declines in mature retinal ganglion cells (RGCs). While a number of transcription factors and signaling molecules have been implicated to the loss of regenerative potential of RGC axon, their upstream regulators are unclear. We investigated the association between developmental decline of RGC regenerative potential and age-related changes in microRNA (miRNA) expression and showed that loss of axon regenerative potential can be partially restored by upregulating miR-19a in RGCs in vitro and in vivo. Regulating miRNA expression represents a new potential therapeutic approach to resuscitate age-related loss of axon growth ability.

Abstract: Axon regeneration capacity declines in mature retinal ganglion cells (RGCs). While a number of transcription factors and signaling molecules have been implicated to the loss of regenerative potential of RGC axon, their upstream regulators are unclear. We investigated the association between developmental decline of RGC regenerative potential and age-related changes in microRNA (miRNA) expression and showed that loss of axon regenerative potential can be partially restored by upregulating miR-19a in RGCs in vitro and in vivo. Regulating miRNA expression represents a new potential therapeutic approach to resuscitate age-related loss of axon growth ability.

Abstract: Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new therapies to promptly restore vision. Innate lymphoid cells (ILCs) are a recently identified heterogeneous cell population that has been reported to orchestrate immunity and promote tissue repair in the lungs and skin after injury. However, whether ILCs can modulate the repair process in the cornea remains poorly understood. We identified a population of cornea-resident group 2 ILCs (ILC2s) in mice that express CD127, T1/ST2, CD90, and cKit. This cell population was relatively rare in corneas at a steady state but increased after corneal epithelial abrasion. Moreover, ILC2s were maintained and expanded locally at a steady state and after wounding. Depletion of this cell population caused a delay in corneal wound healing, whereas supplementation of ILC2s through adoptive transfer partially restored the healing process. Further investigation revealed that IL-25, IL-33, and thymic stromal lymphopoietin had critical roles in corneal ILC2 responses and that CCR2- corneal macrophages were an important producer of IL-33 in the cornea. Together, these results reveal the critical role of cornea-resident ILC2s in the restoration of corneal epithelial integrity after acute injury and suggest that ILC2 responses depend on local induction of IL-25, IL-33, and thymic stromal lymphopoietin.

Abstract: Corneal injuries and infections are the leading cause of blindness worldwide. Thus, understanding the mechanisms that control healing of the damaged cornea is critical for the development of new therapies to promptly restore vision. Innate lymphoid cells (ILCs) are a recently identified heterogeneous cell population that has been reported to orchestrate immunity and promote tissue repair in the lungs and skin after injury. However, whether ILCs can modulate the repair process in the cornea remains poorly understood. We identified a population of cornea-resident group 2 ILCs (ILC2s) in mice that express CD127, T1/ST2, CD90, and cKit. This cell population was relatively rare in corneas at a steady state but increased after corneal epithelial abrasion. Moreover, ILC2s were maintained and expanded locally at a steady state and after wounding. Depletion of this cell population caused a delay in corneal wound healing, whereas supplementation of ILC2s through adoptive transfer partially restored the healing process. Further investigation revealed that IL-25, IL-33, and thymic stromal lymphopoietin had critical roles in corneal ILC2 responses and that CCR2- corneal macrophages were an important producer of IL-33 in the cornea. Together, these results reveal the critical role of cornea-resident ILC2s in the restoration of corneal epithelial integrity after acute injury and suggest that ILC2 responses depend on local induction of IL-25, IL-33, and thymic stromal lymphopoietin.