|
1
|
Iswarya R, Krishnadas S, Dharmalingam K, Gowri Priya C. Human trabecular meshwork stem cell-derived small extracellular vesicles enhance trabecular meshwork cell survival and proliferation. Exp Eye Res 2025; 253:110281. [PMID: 39961413 DOI: 10.1016/j.exer.2025.110281] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2024] [Revised: 02/07/2025] [Accepted: 02/11/2025] [Indexed: 02/21/2025]
Abstract
Glaucoma is an optic neuropathy, one of the leading causes of irreversible blindness worldwide. Previous studies in animal models have shown that transplantation of trabecular meshwork stem cells (TMSCs-adult tissue-resident stem cells of TM) promotes TM regeneration and restores intraocular pressure through paracrine signaling. One of the major paracrine signal mediators is the extracellular vesicles. Given the advantages of sEV over cell-based therapies, the current work aims to investigate the potential of TMSC-derived small extracellular vesicles (sEV) in promoting TM cell survival and proliferation using in vitro experiments. TM cells were cultured in TM media and stem cell growth media (SCGM). Phenotypic and functional (sphere formation) characterization of cultured cells revealed that the SCGM maintained stemness with greater functional efficacy. sEV from TM cell (TM media) and TMSC (SCGM) conditioned media were isolated using the ultracentrifugation method. Characterization of sEV demonstrated that the sEV were within the size range of 30-200 nm and poly-dispersive spherical in shape. The TM and TMSC sEV express common exosomal marker syntenin, TM specific exosomal markers-emilin and neuropilin. To check the uptake specificity, the labelled sEV were incubated with different cell types. The varying degrees of uptake of the labelled sEV by TM cells, HLEB3 and 3T3 cell lines implied that TM and TMSC sEV might have varied surface components. The regenerative efficacy of the sEV was assessed in vitro by scratch wound assay, immunostaining for proliferation marker Ki67, and 5'-Bromo-2'-deoxyuridine incorporation assay. The TMSC sEV exhibited better wound healing efficacy by inducing TM cell proliferation. Furthermore, evaluation of the antioxidant potential depicted that the TMSC sEV enhanced TM cell viability under chronic oxidative stress by significantly reducing the intracellular reactive oxygen species. Taken together, our study demonstrated for the first time that the TMSC sEV enhanced TM cell proliferation as well as migration in vitro and attenuated oxidative stress-induced cell death by reducing intracellular reactive oxygen species. Further studies in animal models will pave the way for the potential application of TMSC sEV in glaucoma treatment.
Collapse
Affiliation(s)
- Radhakrishnan Iswarya
- Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Madurai, Tamil Nadu, India; Department of Biotechnology, Aravind Medical Research Foundation, Alagappa University, Karaikudi, Tamil Nadu, India
| | - Subbaiah Krishnadas
- Glaucoma Services, Aravind Eye Hospital and Post Graduate Institute of Ophthalmology, Madurai, Tamil Nadu, India
| | - Kuppamuthu Dharmalingam
- Department of Biotechnology, Aravind Medical Research Foundation, Alagappa University, Karaikudi, Tamil Nadu, India; Department of Proteomics, Aravind Medical Research Foundation, Madurai, Tamil Nadu, India
| | - Chidambaranathan Gowri Priya
- Department of Immunology and Stem Cell Biology, Aravind Medical Research Foundation, Madurai, Tamil Nadu, India; Department of Biotechnology, Aravind Medical Research Foundation, Alagappa University, Karaikudi, Tamil Nadu, India.
| |
Collapse
|
|
2
|
Enkhbat M, Mehta JS, Peh GSL, Yim EKF. Biomaterial-based strategies for primary human corneal endothelial cells for therapeutic applications: from cell expansion to transplantable carrier. Biomater Sci 2025; 13:1114-1130. [PMID: 39831824 DOI: 10.1039/d4bm00941j] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2025]
Abstract
The treatment of corneal blindness due to corneal diseases and injuries often requires the transplantation of healthy cadaveric corneal endothelial graft tissue to restore corneal clarity and visual function. However, the limited availability of donor corneas poses a significant challenge in meeting the demand for corneal transplantation. As a result, there is a growing interest in developing strategies alleviate this unmet need, and one of the postulated approaches is to isolate and expand primary human corneal endothelial cells (HCECs) in vitro for use in cell therapy. This review summarizes the recent advancements in the expansion of HCECs using biomaterials. Two principal biomaterial-based approaches, including extracellular matrix (ECM) coating and functionalized synthetic polymers, have been investigated to create an optimal microenvironment for the expansion and maintenance of corneal endothelial cells (CECs). This review highlights the challenges and opportunities in expanding primary HCECs using biomaterials. It emphasizes the importance of optimizing biomaterial properties, cell culture conditions, and the roles of biophysical cues to achieve efficient expansion and functional maintenance of CECs. Biomaterial-based strategies hold significant promise for expanding primary HCECs and improving the outcomes of CEC transplantation. The integration of biomaterials as cell culture substrates and transplantable carriers offers a comprehensive approach to address the limitations associated with current corneal tissue engineering techniques.
Collapse
Affiliation(s)
- Myagmartsend Enkhbat
- Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
| | - Jodhbir S Mehta
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
- Corneal & External Eye Disease Department, Singapore National Eye Centre, Singapore 168751, Singapore
- Singhealth Duke-NUS Ophthalmology & Visual Sciences Academic Clinical Programme, Duke-NUS Graduate Medical School, Singapore 169857, Singapore
| | - Gary S L Peh
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore 169856, Singapore.
- Singhealth Duke-NUS Ophthalmology & Visual Sciences Academic Clinical Programme, Duke-NUS Graduate Medical School, Singapore 169857, Singapore
| | - Evelyn K F Yim
- Department of Chemical Engineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada.
- Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
- Center for Biotechnology and Bioengineering, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada
| |
Collapse
|
|
3
|
Du Y, Bammidi S, Yang E. Trabecular Meshwork Stem Cells for Glaucoma Treatment. Methods Mol Biol 2025; 2858:143-158. [PMID: 39433674 PMCID: PMC11971977 DOI: 10.1007/978-1-0716-4140-8_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2024]
Abstract
Elevated intraocular pressure (IOP) is the most important risk factor for primary open-angle glaucoma (POAG) and currently is the only effective treatment target for glaucoma to prevent vision loss. In POAG patients, the trabecular meshwork (TM) cellularity is reduced which might be the main pathologic reason for the conventional outflow pathway dysfunction leading to elevated IOP. Stem cell-based therapy has been shown promising to reduce IOP and preserve retinal ganglion cells and their function in animal models. In this chapter, we describe the method details on TM stem cell cultivation and identification; induction for differentiation into different cell types, including differentiation to TM cell responsiveness to dexamethasone treatment with phagocytic function; and transplantation into mouse anterior chamber for therapeutic purposes.
Collapse
Affiliation(s)
- Yiqin Du
- Department of Ophthalmology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA.
| | - Sridhar Bammidi
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Enzhi Yang
- Department of Ophthalmology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
| |
Collapse
|
|
4
|
Ciociola EC, Fernandez E, Kaufmann M, Klifto MR. Future directions of glaucoma treatment: emerging gene, neuroprotection, nanomedicine, stem cell, and vascular therapies. Curr Opin Ophthalmol 2024; 35:89-96. [PMID: 37910173 DOI: 10.1097/icu.0000000000001016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/03/2023]
Abstract
PURPOSE OF REVIEW The aim of this article is to summarize current research on novel gene, stem cell, neuroprotective, nanomedicine, and vascular therapies for glaucoma. RECENT FINDINGS Gene therapy using viral vectors and siRNA have been shown to reduce intraocular pressure by altering outflow and production of aqueous humor, to reduce postsurgical fibrosis with few adverse effects, and to increase retinal ganglion cell (RGC) survival in animal studies. Stem cells may treat glaucoma by replacing or stimulating proliferation of trabecular meshwork cells, thus restoring outflow facility. Stem cells can also serve a neuroprotective effect by differentiating into RGCs or preventing RGC loss via secretion of growth factors. Other developing neuroprotective glaucoma treatments which can prevent RGC death include nicotinamide, the NT-501 implant which secretes ciliary neurotrophic factor, and a Fas-L inhibitor which are now being tested in clinical trials. Recent studies on vascular therapy for glaucoma have focused on the ability of Rho Kinase inhibitors and dronabinol to increase ocular blood flow. SUMMARY Many novel stem cell, gene, neuroprotective, nanomedicine, and vascular therapies have shown promise in preclinical studies, but further clinical trials are needed to demonstrate safety and efficacy in human glaucomatous eyes. Although likely many years off, future glaucoma therapy may take a multifaceted approach.
Collapse
Affiliation(s)
| | | | | | - Meredith R Klifto
- Department of Ophthalmology, University of North Carolina, Chapel Hill, North Carolina, USA
| |
Collapse
|
|
5
|
Xiao Y, McGhee CNJ, Zhang J. Adult stem cells in the eye: Identification, characterisation, and therapeutic application in ocular regeneration - A review. Clin Exp Ophthalmol 2024; 52:148-166. [PMID: 38214071 DOI: 10.1111/ceo.14309] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Accepted: 09/25/2023] [Indexed: 01/13/2024]
Abstract
Adult stem cells, present in various parts of the human body, are undifferentiated cells that can proliferate and differentiate to replace dying cells within tissues. Stem cells have specifically been identified in the cornea, trabecular meshwork, crystalline lens, iris, ciliary body, retina, choroid, sclera, conjunctiva, eyelid, lacrimal gland, and orbital fat. The identification of ocular stem cells broadens the potential therapeutic strategies for untreatable eye diseases. Currently, stem cell transplantation for corneal and conjunctival diseases remains the most common stem cell-based therapy in ocular clinical management. Lens epithelial stem cells have been applied in the treatment of paediatric cataracts. Several early-phase clinical trials for corneal and retinal regeneration using ocular stem cells are also underway. Extensive preclinical studies using ocular stem cells have been conducted, showing encouraging outcomes. Ocular stem cells currently demonstrate great promise in potential treatments of eye diseases. In this review, we focus on the identification, characterisation, and therapeutic application of adult stem cells in the eye.
Collapse
Affiliation(s)
- Yuting Xiao
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
| | - Charles N J McGhee
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
| | - Jie Zhang
- Department of Ophthalmology, New Zealand National Eye Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
| |
Collapse
|
|
6
|
Buffault J, Brignole-Baudouin F, Labbé A, Baudouin C. An Overview of Current Glaucomatous Trabecular Meshwork Models. Curr Eye Res 2023; 48:1089-1099. [PMID: 37661784 DOI: 10.1080/02713683.2023.2253378] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2023] [Revised: 07/26/2023] [Accepted: 08/25/2023] [Indexed: 09/05/2023]
Abstract
PURPOSE To provide an overview of the existing alternative models for studying trabecular meshwork (TM). METHODS Literature review. RESULTS The TM is a complex tissue that regulates aqueous humor outflow from the eye. Dysfunction of the TM is a major contributor to the pathogenesis of open-angle glaucoma, a leading cause of irreversible blindness worldwide. The TM is a porous structure composed of trabecular meshwork cells (TMC) within a multi-layered extracellular matrix (ECM). Although dysregulation of the outflow throughout the TM represents the first step in the disease process, the underlying mechanisms of TM degeneration associate cell loss and accumulation of ECM, but remain incompletely understood, and drugs targeting the TM are limited. Therefore, experimental models of glaucomatous trabeculopathy are necessary for preclinical screening, to advance research on this disease's pathophysiology, and to develop new therapeutic strategies targeting the TM. Traditional animal models have been used extensively, albeit with inherent limitations, including ethical concerns and limited translatability to humans. Consequently, there has been an increasing focus on developing alternative in vitro models to study the TM. Recent advancements in three-dimensional cell culture and tissue engineering are still in their early stages and do not yet fully reflect the complexity of the outflow pathway. However, they have shown promise in reducing reliance on animal experimentation in certain aspects of glaucoma research. CONCLUSION This review provides an overview of the existing alternative models for studying TM and their potential for advancing research on the pathophysiology of open-angle glaucoma and developing new therapeutic strategies.
Collapse
Affiliation(s)
- Juliette Buffault
- Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, IHU Foresight, Paris, France
- Institut de la Vision, Sorbonne Université, INSERM, CNRS, IHU Foresight, Paris, France
- Department of Ophthalmology, Ambroise Paré Hospital, APHP, Université de Versailles Saint-Quentin-en-Yvelines, Boulogne-Billancourt, France
| | - Françoise Brignole-Baudouin
- Institut de la Vision, Sorbonne Université, INSERM, CNRS, IHU Foresight, Paris, France
- Department of Biology, Quinze-Vingts National Ophthalmology Hospital, IHU Foresight, Paris, France
| | - Antoine Labbé
- Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, IHU Foresight, Paris, France
- Institut de la Vision, Sorbonne Université, INSERM, CNRS, IHU Foresight, Paris, France
- Department of Ophthalmology, Ambroise Paré Hospital, APHP, Université de Versailles Saint-Quentin-en-Yvelines, Boulogne-Billancourt, France
| | - Christophe Baudouin
- Department of Ophthalmology III, Quinze-Vingts National Ophthalmology Hospital, IHU Foresight, Paris, France
- Institut de la Vision, Sorbonne Université, INSERM, CNRS, IHU Foresight, Paris, France
| |
Collapse
|
|
7
|
Yam GHF, Pi S, Du Y, Mehta JS. Posterior corneoscleral limbus: Architecture, stem cells, and clinical implications. Prog Retin Eye Res 2023; 96:101192. [PMID: 37392960 DOI: 10.1016/j.preteyeres.2023.101192] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2023] [Revised: 06/22/2023] [Accepted: 06/23/2023] [Indexed: 07/03/2023]
Abstract
The limbus is a transition from the cornea to conjunctiva and sclera. In human eyes, this thin strip has a rich variation of tissue structures and composition, typifying a change from scleral irregularity and opacity to corneal regularity and transparency; a variation from richly vascularized conjunctiva and sclera to avascular cornea; the neural passage and drainage of aqueous humor. The limbal stroma is enriched with circular fibres running parallel to the corneal circumference, giving its unique role in absorbing small pressure changes to maintain corneal curvature and refractivity. It contains specific niches housing different types of stem cells for the corneal epithelium, stromal keratocytes, corneal endothelium, and trabecular meshwork. This truly reflects the important roles of the limbus in ocular physiology, and the limbal functionality is crucial for corneal health and the entire visual system. Since the anterior limbus containing epithelial structures and limbal epithelial stem cells has been extensively reviewed, this article is focused on the posterior limbus. We have discussed the structural organization and cellular components of the region beneath the limbal epithelium, the characteristics of stem cell types: namely corneal stromal stem cells, endothelial progenitors and trabecular meshwork stem cells, and recent advances leading to the emergence of potential cell therapy options to replenish their respective mature cell types and to correct defects causing corneal abnormalities. We have reviewed different clinical disorders associated with defects of the posterior limbus and summarized the available preclinical and clinical evidence about the developing topic of cell-based therapy for corneal disorders.
Collapse
Affiliation(s)
- Gary Hin-Fai Yam
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA; Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore; McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA.
| | - Shaohua Pi
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA, USA
| | - Yiqin Du
- Department of Ophthalmology, University of South Florida, Tampa, FL, USA
| | - Jodhbir S Mehta
- Tissue Engineering and Cell Therapy Group, Singapore Eye Research Institute, Singapore; Department of Cornea and External Eye Disease, Singapore National Eye Centre, Singapore; Ophthalmology and Visual Sciences Academic Clinical Program, Duke-National University of Singapore (NUS) Medical School, Singapore.
| |
Collapse
|
|
8
|
Abstract
The trabecular meshwork (TM) of the eye serves as an essential tissue in controlling aqueous humor (AH) outflow and intraocular pressure (IOP) homeostasis. However, dysfunctional TM cells and/or decreased TM cellularity is become a critical pathogenic cause for primary open-angle glaucoma (POAG). Consequently, it is particularly valuable to investigate TM characteristics, which, in turn, facilitates the development of new treatments for POAG. Since 2006, the advancement in induced pluripotent stem cells (iPSCs) provides a new tool to (1) model the TM in vitro and (2) regenerate degenerative TM in POAG. In this context, we first summarize the current approaches to induce the differentiation of TM-like cells from iPSCs and compare iPSC-derived TM models to the conventional in vitro TM models. The efficacy of iPSC-derived TM cells for TM regeneration in POAG models is also discussed. Through these approaches, iPSCs are becoming essential tools in glaucoma modeling and for developing personalized treatments for TM regeneration.
Collapse
Affiliation(s)
- Wei Zhu
- Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China.
- Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beihang University & Capital Medical University, Beijing, China.
| | - Xiaoyan Zhang
- Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China
| | - Shen Wu
- Beijing Institute of Ophthalmology, Beijing Tongren Hospital Eye Center, Capital Medical University, Beijing, China
| | - Ningli Wang
- Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beihang University & Capital Medical University, Beijing, China
- Beijing Institute of Ophthalmology, Beijing Tongren Hospital Eye Center, Capital Medical University, Beijing, China
| | - Markus H Kuehn
- Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA
- Center for the Prevention and Treatment of Visual Loss, Iowa City Veterans Affairs Medical Center, Iowa City, IA, USA
| |
Collapse
|
|
9
|
Kopecny LR, Lee BWH, Coroneo MT. A systematic review on the effects of ROCK inhibitors on proliferation and/or differentiation in human somatic stem cells: A hypothesis that ROCK inhibitors support corneal endothelial healing via acting on the limbal stem cell niche. Ocul Surf 2023; 27:16-29. [PMID: 36586668 DOI: 10.1016/j.jtos.2022.12.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2022] [Revised: 12/18/2022] [Accepted: 12/22/2022] [Indexed: 12/29/2022]
Abstract
Rho kinase inhibitors (ROCKi) have attracted growing multidisciplinary interest, particularly in Ophthalmology where the question as to how they promote corneal endothelial healing remains unresolved. Concurrently, stem cell biology has rapidly progressed in unravelling drivers of stem cell (SC) proliferation and differentiation, where mechanical niche factors and the actin cytoskeleton are increasingly recognized as key players. There is mounting evidence from the study of the peripheral corneal endothelium that supports the likelihood of an internal limbal stem cell niche. The possibility that ROCKi stimulate the endothelial SC niche has not been addressed. Furthermore, there is currently a paucity of data that directly evaluates whether ROCKi promotes corneal endothelial healing by acting on this limbal SC niche located near the transition zone. Therefore, we performed a systematic review examining the effects ROCKi on the proliferation and differentiation of human somatic SC, to provide insight into its effects on various human SC populations. An appraisal of electronic searches of four databases identified 1 in vivo and 58 in vitro studies (36 evaluated proliferation while 53 examined differentiation). Types of SC studied included mesenchymal (n = 32), epithelial (n = 11), epidermal (n = 8), hematopoietic and other (n = 8). The ROCK 1/2 selective inhibitor Y-27632 was used in almost all studies (n = 58), while several studies evaluated ≥2 ROCKi (n = 4) including fasudil, H-1152, and KD025. ROCKi significantly influenced human somatic SC proliferation in 81% of studies (29/36) and SC differentiation in 94% of studies (50/53). The present systemic review highlights that ROCKi are influential in regulating human SC proliferation and differentiation, and provides evidence to support the hypothesis that ROCKi promotes corneal endothelial division and maintenance via acting on the inner limbal SC niche.
Collapse
Affiliation(s)
- Lloyd R Kopecny
- School of Clinical Medicine, University of New South Wales, Sydney, Australia.
| | - Brendon W H Lee
- Department of Ophthalmology, School of Clinical Medicine, University of New South Wales, Level 2 South Wing, Edmund Blacket Building, Prince of Wales Hospital, Randwick, NSW, 2031, Australia
| | - Minas T Coroneo
- Department of Ophthalmology, Prince of Wales Hospital, Sydney, Australia
| |
Collapse
|
|
10
|
Autophagy Mediates MMP-2 Expression in Glaucomatous Trabecular Meshwork Cells. J Ophthalmol 2022; 2022:6026464. [PMID: 36211598 PMCID: PMC9536984 DOI: 10.1155/2022/6026464] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2022] [Accepted: 07/25/2022] [Indexed: 11/24/2022] Open
Abstract
Purpose To investigate the effect of 3-methyladenine (3-MA) and starvation on the expression of matrix metalloproteinase (MMP-2) in patients with primary open-angle glaucoma. Methods Primary TM cells were cultured and divided into three groups. The control group was treated with a normal medium, the 3-MA group was stimulated with 3-MA, and the starvation group received nutrient depletion by replacing the normal media with Earle's balanced salt solution. Cellular mRNA and protein were measured at different 3-MA concentrations and starvation time periods. The level of autophagy was accessed by monodansylcadaverine fluorescent staining and expression of specific autophagy-related genes, light chain 3 (LC3), and Beclin1. The effects of 3-MA and starvation on cell proliferation were determined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay kit. The mRNA and protein expression of LC3-II, Beclin1, and MMP-2 were measured by reverse transcription-polymerase chain reaction and western blot, respectively. Results Compared to the control group, starvation significantly upregulated LC3-II and Beclin1 in TM cells after 3 h of stimulation, which peaked at 6 h and 9 h, respectively. Increased MDC-labeled cells were also observed. Starvation downregulated the expression of MMP-2. On the contrary, 3-MA suppressed the activation of autophagy, as shown by the marked downregulation of LC3-II and Beclin1. The expressions of MMP-2 were higher in the 3-MA group compared to the control group, reaching a peak at a concentration of 5 mM. Conclusion Autophagy may be involved in the pathogenesis of POAG via regulating the expression of MMP-2 and, subsequently, the deposition of the extracellular matrix.
Collapse
|
|
11
|
Coulon SJ, Schuman JS, Du Y, Bahrani Fard MR, Ethier CR, Stamer WD. A novel glaucoma approach: Stem cell regeneration of the trabecular meshwork. Prog Retin Eye Res 2022; 90:101063. [PMID: 35398015 PMCID: PMC9464663 DOI: 10.1016/j.preteyeres.2022.101063] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2021] [Revised: 03/20/2022] [Accepted: 03/24/2022] [Indexed: 12/13/2022]
Abstract
Glaucoma is the leading cause of global irreversible blindness, necessitating research for new, more efficacious treatment options than currently exist. Trabecular meshwork (TM) cells play an important role in the maintenance and function of the aqueous outflow pathway, and studies have found that there is decreased cellularity of the TM in glaucoma. Regeneration of the TM with stem cells has been proposed as a novel therapeutic option by several reports over the last few decades. Stem cells have the capacity for self-renewal and the potential to differentiate into adult functional cells. Several types of stem cells have been investigated in ocular regenerative medicine: tissue specific stem cells, embryonic stem cells, induced pluripotent stem cells, and adult mesenchymal stem cells. These cells have been used in various glaucoma animal models and ex vivo models and have shown success in IOP homeostasis and TM cellularity restoration. They have also demonstrated stability without serious side effects for a significant period of time. Based on current knowledge of TM pathology in glaucoma and existing literature regarding stem cell regeneration of this tissue, we propose a human clinical study as the next step in understanding this potentially revolutionary treatment paradigm. The ability to protect and replace TM cells in glaucomatous eyes could change the field forever.
Collapse
Affiliation(s)
- Sara J Coulon
- Department of Ophthalmology, NYU Grossman School of Medicine, NYU Langone Health, New York University, New York, NY, USA
| | - Joel S Schuman
- Department of Ophthalmology, NYU Grossman School of Medicine, NYU Langone Health, New York University, New York, NY, USA; Center for Neural Science, College of Arts and Science, New York University, New York, NY, USA; Departments of Biomedical Engineering and Electrical and Computer Engineering, New York University Tandon School of Engineering, Brooklyn, NY, USA; Department of Physiology and Neuroscience, NYU Grossman School of Medicine, NYU Langone Health, New York University, New York, NY, USA.
| | - Yiqin Du
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Mohammad Reza Bahrani Fard
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University School of Medicine, Atlanta, GA, USA
| | - C Ross Ethier
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology & Emory University School of Medicine, Atlanta, GA, USA
| | - W Daniel Stamer
- Departments of Ophthalmology and Biomedical Engineering, Duke University, Durham, NC, USA
| |
Collapse
|
|
12
|
Murphy-Ullrich JE. Thrombospondin-1 Signaling Through the Calreticulin/LDL Receptor Related Protein 1 Axis: Functions and Possible Roles in Glaucoma. Front Cell Dev Biol 2022; 10:898772. [PMID: 35693935 PMCID: PMC9185677 DOI: 10.3389/fcell.2022.898772] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Accepted: 05/10/2022] [Indexed: 11/13/2022] Open
Abstract
Thrombospondin-1 (TSP-1) is a matricellular extracellular matrix protein. Matricellular proteins are components of the extracellular matrix (ECM) that regulate key cellular functions and impact ECM organization, but which lack direct primary structural roles in the ECM. TSP-1 expression is upregulated in response to injury, hypoxia, growth factor stimulation, inflammation, glucose, and by reactive oxygen species. Relevant to glaucoma, TSP-1 is also a mechanosensitive molecule upregulated by mechanical stretch. TSP-1 expression is increased in ocular remodeling in glaucoma in both the trabecular meshwork and in the optic nerve head. The exact roles of TSP-1 in glaucoma remain to be defined, however. It plays important roles in cell behavior and in ECM remodeling during wound healing, fibrosis, angiogenesis, and in tumorigenesis and metastasis. At the cellular level, TSP-1 can modulate cell adhesion and migration, protease activity, growth factor activity, anoikis resistance, apoptosis, and collagen secretion and matrix assembly and cross-linking. These multiple functions and macromolecular and receptor interactions have been ascribed to specific domains of the TSP-1 molecule. In this review, we will focus on the cell regulatory activities of the TSP-1 N-terminal domain (NTD) sequence that binds to cell surface calreticulin (Calr) and which regulates cell functions via signaling through Calr complexed with LDL receptor related protein 1 (LRP1). We will describe TSP-1 actions mediated through the Calr/LRP1 complex in regulating focal adhesion disassembly and cytoskeletal reorganization, cell motility, anoikis resistance, and induction of collagen secretion and matrix deposition. Finally, we will consider the relevance of these TSP-1 functions to the pathologic remodeling of the ECM in glaucoma.
Collapse
Affiliation(s)
- Joanne E. Murphy-Ullrich
- Departments of Pathology, University of Alabama at Birmingham, Birmingham, AL, United States
- Department of Cell Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, United States
- Department of Ophthalmology and Visual Sciences, University of Alabama at Birmingham, Birmingham, AL, United States
- *Correspondence: Joanne E. Murphy-Ullrich,
| |
Collapse
|
|
13
|
Wang X, Cao Q, Wu S, Bahrani Fard MR, Wang N, Cao J, Zhu W. Magnetic Nano-Platform Enhanced iPSC-Derived Trabecular Meshwork Delivery and Tracking Efficiency. Int J Nanomedicine 2022; 17:1285-1307. [PMID: 35345785 PMCID: PMC8957401 DOI: 10.2147/ijn.s346141] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Accepted: 03/09/2022] [Indexed: 11/23/2022] Open
Abstract
Purpose Transplantation of stem cells to remodel the trabecular meshwork (TM) has become a new option for restoring aqueous humor dynamics and intraocular pressure homeostasis in glaucoma. In this study, we aimed to design a nanoparticle to label induced pluripotent stem cell (iPSC)-derived TM and improve the delivery accuracy and in vivo tracking efficiency. Methods PLGA-SPIO-Cypate (PSC) NPs were designed with polylactic acid-glycolic acid (PLGA) polymers as the backbone, superparamagnetic iron oxide (SPIO) nanoparticles, and near-infrared (NIR) dye cypate. In vitro assessment of cytotoxicity, iron content after NPs labeling, and the dual-model monitor was performed on mouse iPSC-derived TM (miPSC-TM) cells, as well as immortalized and primary human TM cells. Cell function after labeling, the delivery accuracy, in vivo tracking efficiency, and its effect on lowering IOP were evaluated following miPSC-TM transplantation in mice. Results Initial in vitro experiments showed that a single-time nanoparticles incubation was sufficient to label iPSC-derived TM and was not related to any change in both cell viability and fate. Subsequent in vivo evaluation revealed that the use of this nanoparticle not only improves the delivery accuracy of the transplanted cells in live animals but also benefits the dual-model tracking in the long term. More importantly, the use of the magnet triggers a temporary enhancement in the effectiveness of cell-based therapy in alleviating the pathologies associated with glaucoma. Conclusion This study provided a promising approach for enhancing both the delivery and in vivo tracking efficiency of the transplanted cells, which facilitates the clinical translation of stem cell-based therapy for glaucoma.
Collapse
Affiliation(s)
- Xiangji Wang
- School of Pharmacy, Qingdao University, Qingdao, People's Republic of China
| | - Qilong Cao
- Qingdao Haier Biotech Co. Ltd, Qingdao, People's Republic of China
| | - Shen Wu
- Beijing Tongren Hospital Eye Center, Capital Medical University, Beijing, People's Republic of China
| | | | - Ningli Wang
- Beijing Tongren Hospital Eye Center, Capital Medical University, Beijing, People's Republic of China
| | - Jie Cao
- School of Pharmacy, Qingdao University, Qingdao, People's Republic of China
| | - Wei Zhu
- School of Pharmacy, Qingdao University, Qingdao, People's Republic of China.,Beijing Advanced Innovation Center for Big Data-Based Precision Medicine, Beihang University & Capital Medical University, Beijing, People's Republic of China
| |
Collapse
|
|
14
|
Dada T, Mahalingam K, Bhartiya S. Minimally Invasive Glaucoma Surgery-to Remove or Preserve the Trabecular Meshwork: That is the Question? J Curr Glaucoma Pract 2021; 15:47-51. [PMID: 34720492 PMCID: PMC8543745 DOI: 10.5005/jp-journals-10078-1299] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
How to cite this article: Dada T, Mahalingam K, Bhartiya S. Minimally Invasive Glaucoma Surgery—to Remove or Preserve the Trabecular Meshwork: That is the Question? J Curr Glaucoma Pract 2021;15(2):47–51.
Collapse
Affiliation(s)
- Tanuj Dada
- Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
| | - Karthikeyan Mahalingam
- Dr Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India
| | - Shibal Bhartiya
- Department of Ophthalmology, Glaucoma Services, Fortis Memorial Hospital, Gurugram, Haryana, India
| |
Collapse
|
|
15
|
Hidalgo-Alvarez V, Dhowre HS, Kingston OA, Sheridan CM, Levis HJ. Biofabrication of Artificial Stem Cell Niches in the Anterior Ocular Segment. Bioengineering (Basel) 2021; 8:135. [PMID: 34677208 PMCID: PMC8533470 DOI: 10.3390/bioengineering8100135] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Revised: 09/27/2021] [Accepted: 09/27/2021] [Indexed: 11/16/2022] Open
Abstract
The anterior segment of the eye is a complex set of structures that collectively act to maintain the integrity of the globe and direct light towards the posteriorly located retina. The eye is exposed to numerous physical and environmental insults such as infection, UV radiation, physical or chemical injuries. Loss of transparency to the cornea or lens (cataract) and dysfunctional regulation of intra ocular pressure (glaucoma) are leading causes of worldwide blindness. Whilst traditional therapeutic approaches can improve vision, their effect often fails to control the multiple pathological events that lead to long-term vision loss. Regenerative medicine approaches in the eye have already had success with ocular stem cell therapy and ex vivo production of cornea and conjunctival tissue for transplant recovering patients' vision. However, advancements are required to increase the efficacy of these as well as develop other ocular cell therapies. One of the most important challenges that determines the success of regenerative approaches is the preservation of the stem cell properties during expansion culture in vitro. To achieve this, the environment must provide the physical, chemical and biological factors that ensure the maintenance of their undifferentiated state, as well as their proliferative capacity. This is likely to be accomplished by replicating the natural stem cell niche in vitro. Due to the complex nature of the cell microenvironment, the creation of such artificial niches requires the use of bioengineering techniques which can replicate the physico-chemical properties and the dynamic cell-extracellular matrix interactions that maintain the stem cell phenotype. This review discusses the progress made in the replication of stem cell niches from the anterior ocular segment by using bioengineering approaches and their therapeutic implications.
Collapse
Affiliation(s)
- Veronica Hidalgo-Alvarez
- Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK
| | - Hala S. Dhowre
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, UK; (H.S.D.); (O.A.K.)
| | - Olivia A. Kingston
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, UK; (H.S.D.); (O.A.K.)
| | - Carl M. Sheridan
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, UK; (H.S.D.); (O.A.K.)
| | - Hannah J. Levis
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L7 8TX, UK; (H.S.D.); (O.A.K.)
| |
Collapse
|
|
16
|
Fan X, Bilir EK, Kingston OA, Oldershaw RA, Kearns VR, Willoughby CE, Sheridan CM. Replacement of the Trabecular Meshwork Cells-A Way Ahead in IOP Control? Biomolecules 2021; 11:biom11091371. [PMID: 34572584 PMCID: PMC8464777 DOI: 10.3390/biom11091371] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2021] [Revised: 09/09/2021] [Accepted: 09/13/2021] [Indexed: 12/11/2022] Open
Abstract
Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM's structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.
Collapse
Affiliation(s)
- Xiaochen Fan
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L69 3BX, UK; (X.F.); (E.K.B.); (O.A.K.); (V.R.K.)
| | - Emine K. Bilir
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L69 3BX, UK; (X.F.); (E.K.B.); (O.A.K.); (V.R.K.)
| | - Olivia A. Kingston
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L69 3BX, UK; (X.F.); (E.K.B.); (O.A.K.); (V.R.K.)
| | - Rachel A. Oldershaw
- Department of Musculoskeletal and Ageing Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L69 3BX, UK;
| | - Victoria R. Kearns
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L69 3BX, UK; (X.F.); (E.K.B.); (O.A.K.); (V.R.K.)
| | - Colin E. Willoughby
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L69 3BX, UK; (X.F.); (E.K.B.); (O.A.K.); (V.R.K.)
- Genomic Medicine, Biomedical Sciences Research Institute, Ulster University, Coleraine BT52 1SA, UK
- Correspondence: (C.E.W.); (C.M.S.); Tel.: +44-(28)-701-2338 (C.E.W.); +44-(151)-794-9031 (C.M.S.)
| | - Carl M. Sheridan
- Department of Eye and Vision Science, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool L69 3BX, UK; (X.F.); (E.K.B.); (O.A.K.); (V.R.K.)
- Correspondence: (C.E.W.); (C.M.S.); Tel.: +44-(28)-701-2338 (C.E.W.); +44-(151)-794-9031 (C.M.S.)
| |
Collapse
|
|
17
|
Cell-Based Therapies for Trabecular Meshwork Regeneration to Treat Glaucoma. Biomolecules 2021; 11:biom11091258. [PMID: 34572471 PMCID: PMC8465897 DOI: 10.3390/biom11091258] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2021] [Accepted: 08/01/2021] [Indexed: 12/23/2022] Open
Abstract
Glaucoma is clinically characterized by elevated intraocular pressure (IOP) that leads to retinal ganglion cell (RGC) and optic nerve damage, and eventually blindness if left untreated. Even in normal pressure glaucoma patients, a reduction of IOP is currently the only effective way to prevent blindness, by either increasing aqueous humor outflow or decreasing aqueous humor production. The trabecular meshwork (TM) and the adjacent Schlemm’s canal inner wall play a key role in regulating IOP by providing resistance when aqueous humor drains through the tissue. TM dysfunction seen in glaucoma, through reduced cellularity, abnormal extracellular matrix accumulation, and increased stiffness, contributes to elevated IOP, but current therapies do not target the TM tissue. Stem cell transplantation for regeneration and re-functionalization of damaged TM has shown promise in providing a more direct and effective therapy for glaucoma. In this review, we describe the use of different types of stem cells for TM regeneration in glaucoma models, the mechanisms of regeneration, and the potential for glaucoma treatment using autologous stem cell transplantation.
Collapse
|
|
18
|
Mannino G, Russo C, Longo A, Anfuso CD, Lupo G, Lo Furno D, Giuffrida R, Giurdanella G. Potential therapeutic applications of mesenchymal stem cells for the treatment of eye diseases. World J Stem Cells 2021; 13:632-644. [PMID: 34249232 PMCID: PMC8246249 DOI: 10.4252/wjsc.v13.i6.632] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 05/07/2021] [Accepted: 06/04/2021] [Indexed: 02/06/2023] Open
Abstract
Stem cell-based treatments have been extensively explored in the last few decades to develop therapeutic strategies aimed at providing effective alternatives for those human pathologies in which surgical or pharmacological therapies produce limited effects. Among stem cells of different sources, mesenchymal stem cells (MSCs) offer several advantages, such as the absence of ethical concerns, easy harvesting, low immunogenicity and reduced tumorigenesis risks. Other than a multipotent differentiation ability, MSCs can release extracellular vesicles conveying proteins, mRNA and microRNA. Thanks to these properties, new therapeutic approaches have been designed for the treatment of various pathologies, including ocular diseases. In this review, the use of different MSCs and different administration strategies are described for the treatment of diabetic retinopathy, glaucoma, and retinitis pigmentosa. In a large number of investigations, positive results have been obtained by in vitro experiments and by MSC administration in animal models. Most authors agree that beneficial effects are likely related to MSC paracrine activity. Based on these considerations, many clinical trials have already been carried out. Overall, although some adverse effects have been described, promising outcomes are reported. It can be assumed that in the near future, safer and more effective protocols will be developed for more numerous clinical applications to improve the quality of life of patients affected by eye diseases.
Collapse
Affiliation(s)
- Giuliana Mannino
- Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
| | - Cristina Russo
- Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
| | - Anna Longo
- Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
| | - Carmelina Daniela Anfuso
- Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
| | - Gabriella Lupo
- Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
| | - Debora Lo Furno
- Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy.
| | - Rosario Giuffrida
- Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
| | - Giovanni Giurdanella
- Department of Biomedical and Biotechnological Sciences, School of Medicine, University of Catania, Catania 95123, Italy
| |
Collapse
|
|
19
|
Wang W, Miao Y, Sui S, Wang Y, Wu S, Cao Q, Duan H, Qi X, Zhou Q, Pan X, Zhang J, Chen X, Han Y, Wang N, Kuehn MH, Zhu W. Xeno- and Feeder-Free Differentiation of Human iPSCs to Trabecular Meshwork-Like Cells by Recombinant Cytokines. Transl Vis Sci Technol 2021; 10:27. [PMID: 34015102 PMCID: PMC8142710 DOI: 10.1167/tvst.10.6.27] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2020] [Accepted: 03/01/2021] [Indexed: 12/22/2022] Open
Abstract
Purpose Stem cell-based therapy has the potential to become one approach to regenerate the damaged trabecular meshwork (TM) in glaucoma. Co-culture of induced pluripotent stem cells (iPSCs) with human TM cells has been a successful approach to generate autologous TM resembling cells. However, the differentiated cells generated using this approach are still problematic for clinical usage. This study aimed to develop a clinically applicable strategy for generating TM-like cells from iPSCs. Methods Highly expressed receptors during iPSC differentiation were identified by AutoSOME, Gene Ontology, and reverse transcription polymerase chain reaction (RT-PCR) analysis. The recombinant cytokines that bind to these receptors were used to generate a new differentiation protocol. The resultant TM-like cells were characterized morphologically, immunohistochemically, and transcriptionally. Results We first determined two stages of iPSC differentiation and identified highly expressed receptors associated with the differentiation at each stage. The expression of these receptors was further confirmed by RT-PCR analysis. Exposure to the recombinant cytokines that bind to these receptors, including transforming growth factor beta 1, nerve growth factor beta, erythropoietin, prostaglandin F2 alpha, and epidermal growth factor, can efficiently differentiate iPSCs into TM-like cells, which express TM biomarkers and can form dexamethasone-inducible CLANs. Conclusions We successfully generated a xeno- and feeder-free differentiation protocol with recombinant cytokines to generate the TM progenitor and TM-like cells from human iPSCs. Translational Relevance The new approach minimizes the risks from contamination and also improves the differentiation efficiency and consistency, which are particularly crucial for clinical use of stem cells in glaucoma treatment.
Collapse
Affiliation(s)
- Wenyan Wang
- Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Yongzhen Miao
- Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China
| | - Shangru Sui
- Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China
| | - Yanan Wang
- Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Shen Wu
- Beijing Institute of Ophthalmology, Beijing Tongren Hospital Eye Center, Beijing, China
| | - Qilong Cao
- Qingdao Haier Biotech Co. Ltd., Qingdao, China
| | - Haoyun Duan
- Qingdao Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China
| | - Xia Qi
- Qingdao Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China
| | - Qingjun Zhou
- Qingdao Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China
| | - Xiaojing Pan
- Qingdao Eye Hospital, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, China
| | - Jingxue Zhang
- Beijing Institute of Ophthalmology, Beijing Tongren Hospital Eye Center, Beijing, China
| | - Xuehong Chen
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Yantao Han
- School of Basic Medicine, Qingdao University, Qingdao, China
| | - Ningli Wang
- Beijing Institute of Ophthalmology, Beijing Tongren Hospital Eye Center, Beijing, China
| | - Markus H. Kuehn
- Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA
- Center for the Prevention and Treatment of Visual Loss, Iowa City Veterans Affairs Medical Center, Iowa City, IA, USA
| | - Wei Zhu
- Department of Pharmacology, School of Pharmacy, Qingdao University, Qingdao, China
- Advanced Innovation Center for Big Data-Based Precision Medicine, Beijing University of Aeronautics and Astronautics-Capital Medical University, Beijing, China
| |
Collapse
|
|
20
|
Xiong S, Kumar A, Tian S, Taher EE, Yang E, Kinchington PR, Xia X, Du Y. Stem cell transplantation rescued a primary open-angle glaucoma mouse model. eLife 2021; 10:63677. [PMID: 33506763 PMCID: PMC7864631 DOI: 10.7554/elife.63677] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Accepted: 01/22/2021] [Indexed: 12/13/2022] Open
Abstract
Glaucoma is a leading cause of irreversible blindness. In this study, we investigated if transplanted stem cells are able to rescue a glaucoma mouse model with transgenic myocilin Y437H mutation and explored the possible mechanisms. Human trabecular meshwork stem cells (TMSCs) were intracamerally transplanted which reduced mouse intraocular pressure, increased outflow facility, protected the retinal ganglion cells and preserved their function. TMSC transplantation also significantly increased the TM cellularity, promoted myocilin secretion from TM cells into the aqueous humor to reduce endoplasmic reticulum stress, repaired the TM tissue with extracellular matrix modulation and ultrastructural restoration. Co-culturing TMSCs with myocilin mutant TM cells in vitro promoted TMSCs differentiating into phagocytic functional TM cells. RNA sequencing revealed that TMSCs had upregulated genes related to TM regeneration and neuroprotection. Our results uncovered therapeutic potential of TMSCs for curing glaucoma and elucidated possible mechanisms by which TMSCs achieve the treatment effect.
Collapse
Affiliation(s)
- Siqi Xiong
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States.,Eye Center of Xiangya Hospital, Central South University, Changsha, China.,Hunan Key Laboratory of Ophthalmology, Changsha, China
| | - Ajay Kumar
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States
| | - Shenghe Tian
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States
| | - Eman E Taher
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States.,Research Institute of Ophthalmology, Giza, Egypt
| | - Enzhi Yang
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States
| | - Paul R Kinchington
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States
| | - Xiaobo Xia
- Eye Center of Xiangya Hospital, Central South University, Changsha, China.,Hunan Key Laboratory of Ophthalmology, Changsha, China
| | - Yiqin Du
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, United States.,Department of Developmental Biology, University of Pittsburgh, Pittsburgh, United States.,McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, United States
| |
Collapse
|
|
21
|
Regenerative capacity of the corneal transition zone for endothelial cell therapy. Stem Cell Res Ther 2020; 11:523. [PMID: 33276809 PMCID: PMC7716425 DOI: 10.1186/s13287-020-02046-2] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2020] [Accepted: 11/23/2020] [Indexed: 12/13/2022] Open
Abstract
The corneal endothelium located on the posterior corneal surface is responsible for regulating stromal hydration. This is contributed by a monolayer of corneal endothelial cells (CECs), which are metabolically active in a continuous fluid-coupled efflux of ions from the corneal stroma into the aqueous humor, preventing stromal over-hydration and preserving the orderly arrangement of stromal collagen fibrils, which is essential for corneal transparency. Mature CECs do not have regenerative capacity and cell loss due to aging and diseases results in irreversible stromal edema and a loss of corneal clarity. The current gold standard of treatment for this worldwide blindness caused by corneal endothelial failure is the corneal transplantation using cadaveric donor corneas. The top indication is Fuchs corneal endothelial dystrophy/degeneration, which represents 39% of all corneal transplants performed. However, the global shortage of transplantable donor corneas has restricted the treatment outcomes, hence instigating a need to research for alternative therapies. One such avenue is the CEC regeneration from endothelial progenitors, which have been identified in the peripheral endothelium and the adjacent transition zone. This review examines the evidence supporting the existence of endothelial progenitors in the posterior limbus and summarizes the existing knowledge on the microanatomy of the transitional zone. We give an overview of the isolation and ex vivo propagation of human endothelial progenitors in the transition zone, and their growth and differentiation capacity to the corneal endothelium. Transplanting these bioengineered constructs into in vivo models of corneal endothelial degeneration will prove the efficacy and viability, and the long-term maintenance of functional endothelium. This will develop a novel regenerative therapy for the management of corneal endothelial diseases.
Collapse
|
|
22
|
Tian YI, Zhang X, Torrejon K, Danias J, Du Y, Xie Y. A Biomimetic, Stem Cell-Derived In Vitro Ocular Outflow Model. ADVANCED BIOSYSTEMS 2020; 4:e2000004. [PMID: 32734694 PMCID: PMC7484422 DOI: 10.1002/adbi.202000004] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/05/2020] [Revised: 06/07/2020] [Indexed: 12/24/2022]
Abstract
Age-related human trabecular meshwork (HTM) cell loss is suggested to affect its ability to regulate aqueous humor outflow in the eye. In addition, disease-related HTM cell loss is suggested to lead to elevated intraocular pressure in glaucoma. Induced pluripotent stem cell (iPSC)-derived trabecular meshwork (TM) cells are promising autologous cell sources that can be used to restore the declining TM cell population and function. Previously, an in vitro HTM model is bioengineered for understanding HTM cell biology and screening of pharmacological or biological agents that affect trabecular outflow facility. In this study, it is demonstrated that human iPSC-derived TM cells cultured on SU-8 scaffolds exhibit HTM-like cell morphology, extracellular matrix deposition, and drug responsiveness to dexamethasone treatment. These findings suggest that iPSC-derived TM cells behave like primary HTM cells and can thus serve as reproducible and scalable cell sources when using this in vitro system for glaucoma drug screening and further understanding of outflow pathway physiology, leading to personalized medicine.
Collapse
Affiliation(s)
- Yangzi Isabel Tian
- College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA
| | - Xulang Zhang
- College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA
| | - Karen Torrejon
- Glauconix Biosciences, Inc., 251 Fuller Road, Albany, NY 12203, USA
| | - John Danias
- SUNY Downstate Medical Center, 450 Clarkson Ave, Brooklyn, NY 11203, USA
| | - Yiqin Du
- University of Pittsburg School of Medicine, 203 Lothrop Street, Pittsburgh, PA 15213, USA
| | - Yubing Xie
- College of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA
| |
Collapse
|
|
23
|
Buffault J, Labbé A, Hamard P, Brignole-Baudouin F, Baudouin C. [The trabecular meshwork: Structure, function and clinical implications. A review of the littérature (French translation of the article)]. J Fr Ophtalmol 2020; 43:779-793. [PMID: 32807552 DOI: 10.1016/j.jfo.2020.04.022] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2020] [Revised: 04/17/2020] [Accepted: 04/22/2020] [Indexed: 11/26/2022]
Abstract
Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, three-dimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.
Collapse
Affiliation(s)
- J Buffault
- Service d'ophtalmologie, centre hospitalier national d'ophtalmologie des Quinze-Vingts, IHU FOReSIGHT, 28, rue de Charenton, 75012 Paris, France.
| | - A Labbé
- Service d'ophtalmologie, centre hospitalier national d'ophtalmologie des Quinze-Vingts, IHU FOReSIGHT, 28, rue de Charenton, 75012 Paris, France; Service d'ophtalmologie, hôpital Ambroise-Paré, AP-HP, 9, avenue Charles-de-Gaulle, 92100 Boulogne-Billancourt, France; Inserm, CNRS, institut de la vision, Sorbonne université, 17, rue Moreau, 75012 Paris, France
| | - P Hamard
- Service d'ophtalmologie, centre hospitalier national d'ophtalmologie des Quinze-Vingts, IHU FOReSIGHT, 28, rue de Charenton, 75012 Paris, France
| | - F Brignole-Baudouin
- Inserm, CNRS, institut de la vision, Sorbonne université, 17, rue Moreau, 75012 Paris, France; Service de biologie médicale, centre hospitalier national d'ophtalmologie des Quinze-Vingts, IHU FOReSIGHT, 28, rue de Charenton, 75012 Paris, France
| | - C Baudouin
- Service d'ophtalmologie, centre hospitalier national d'ophtalmologie des Quinze-Vingts, IHU FOReSIGHT, 28, rue de Charenton, 75012 Paris, France; Service d'ophtalmologie, hôpital Ambroise-Paré, AP-HP, 9, avenue Charles-de-Gaulle, 92100 Boulogne-Billancourt, France; Inserm, CNRS, institut de la vision, Sorbonne université, 17, rue Moreau, 75012 Paris, France
| |
Collapse
|
|
24
|
Kaufman PL. Deconstructing aqueous humor outflow - The last 50 years. Exp Eye Res 2020; 197:108105. [PMID: 32590004 PMCID: PMC7990028 DOI: 10.1016/j.exer.2020.108105] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2020] [Revised: 05/29/2020] [Accepted: 06/02/2020] [Indexed: 12/27/2022]
Abstract
Herein partially summarizes one scientist-clinician's wanderings through the jungles of primate aqueous humor outflow over the past ~45 years. Totally removing the iris has no effect on outflow facility or its response to pilocarpine, whereas disinserting the ciliary muscle (CM) from the scleral spur/trabecular meshwork (TM) completely abolishes pilocarpine's effect. Epinephrine increases facility in CM disinserted eyes. Cytochalasins and latrunculins increase outflow facility, subthreshold doses of cytochalasins and epinephrine given together increase facility, and phalloidin, which has no effect on facility, partially blocks the effect of both cytochalasins and epinephrine. H-7, ML7, Y27632 and nitric oxide - donating compounds all increase facility, consistent with a mechanosensitive TM/SC. Adenosine A1 agonists increase and angiotensin II decrease facility. OCT and optical imaging techniques now permit visualization and digital recording of the distal outflow pathways in real time. Prostaglandin (PG) F2α analogues increase the synthesis and release of matrix metalloproteinases by the CM cells, causing remodeling and thinning of the interbundle extracellular matrix (ECM), thereby increasing uveoscleral outflow and reducing IOP. Combination molecules (one molecule, two or more effects) and fixed combination products (two molecules in one bottle) simplify drug regimens for patients. Gene and stem cell therapies to enhance aqueous outflow have been successful in laboratory models and may fill an unmet need in terms of patient compliance, taking the patient out of the delivery system. Functional transfer of genes inhibiting the rho cascade or decoupling actin from myosin increase facility, while genes preferentially expressed in the glaucomatous TM decrease facility. In live NHP, reporter genes are expressed for 2+ years in the TM after a single intracameral injection, with no adverse reaction. However, except for one recent report, injection of facility-effective genes in monkey organ cultured anterior segments (MOCAS) have no effect in live NHP. While intracameral injection of an FIV. BOVPGFS-myc.GFP PGF synthase vector construct reproducibly induces an ~2 mmHg reduction in IOP, the effect is much less than that of topical PGF2⍺ analogue eyedrops, and dissipates after 5 months. The turnoff mechanism has yet to be defeated, although proteasome inhibition enhances reporter gene expression in MOCAS. Intracanalicular injection might minimize off-target effects that activate turn-off mechanisms. An AD-P21 vector injected sub-tenon is effective in 'right-timing' wound healing after trabeculectomy in live laser-induced glaucomatous monkeys. In human (H)OCAS, depletion of TM cells by saponification eliminates the aqueous flow response to pressure elevation, which can be restored by either cultured TM cells or by IPSC-derived TM cells. There were many other steps along the way, but much was accomplished, biologically and therapeutically over the past half century of research and development focused on one very small but complex ocular apparatus. I am deeply grateful for this award, named for a giant in our field that none of us can live up to.
Collapse
Affiliation(s)
- Paul L Kaufman
- University of Wisconsin - Madison, School of Medicine & Public Health, Dept of Ophthalmology & Visual Sciences, United States.
| |
Collapse
|
|
25
|
The trabecular meshwork: Structure, function and clinical implications. A review of the literature. J Fr Ophtalmol 2020; 43:e217-e230. [PMID: 32561029 DOI: 10.1016/j.jfo.2020.05.002] [Citation(s) in RCA: 72] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2020] [Revised: 04/25/2020] [Accepted: 05/27/2020] [Indexed: 02/06/2023]
Abstract
Glaucoma is a blinding optic neuropathy, the main risk factor for which is increased intraocular pressure (IOP). The trabecular meshwork, located within the iridocorneal angle, is the main pathway for drainage of aqueous humor (AH) out of the eye, and its dysfunction is responsible for the IOP elevation. The trabecular meshwork is a complex, fenestrated, three-dimensional structure composed of trabecular meshwork cells (TMC) interdigitated into a multilayered organization within the extracellular matrix (ECM). The purpose of this literature review is to provide an overview of current understanding of the trabecular meshwork and its pathophysiology in glaucoma. Thus, we will present the main anatomical and cellular bases for the regulation of aqueous humor outflow resistance, the pathophysiological mechanisms involved in trabecular dysfunction in the various types of glaucoma, as well as current and future therapeutic strategies targeting the trabecular meshwork.
Collapse
|
|
26
|
van Zyl T, Yan W, McAdams A, Peng YR, Shekhar K, Regev A, Juric D, Sanes JR. Cell atlas of aqueous humor outflow pathways in eyes of humans and four model species provides insight into glaucoma pathogenesis. Proc Natl Acad Sci U S A 2020; 117:10339-10349. [PMID: 32341164 PMCID: PMC7229661 DOI: 10.1073/pnas.2001250117] [Citation(s) in RCA: 131] [Impact Index Per Article: 26.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Increased intraocular pressure (IOP) represents a major risk factor for glaucoma, a prevalent eye disease characterized by death of retinal ganglion cells; lowering IOP is the only proven treatment strategy to delay disease progression. The main determinant of IOP is the equilibrium between production and drainage of aqueous humor, with compromised drainage generally viewed as the primary contributor to dangerous IOP elevations. Drainage occurs through two pathways in the anterior segment of the eye called conventional and uveoscleral. To gain insights into the cell types that comprise these pathways, we used high-throughput single-cell RNA sequencing (scRNAseq). From ∼24,000 single-cell transcriptomes, we identified 19 cell types with molecular markers for each and used histological methods to localize each type. We then performed similar analyses on four organisms used for experimental studies of IOP dynamics and glaucoma: cynomolgus macaque (Macaca fascicularis), rhesus macaque (Macaca mulatta), pig (Sus scrofa), and mouse (Mus musculus). Many human cell types had counterparts in these models, but differences in cell types and gene expression were evident. Finally, we identified the cell types that express genes implicated in glaucoma in all five species. Together, our results provide foundations for investigating the pathogenesis of glaucoma and for using model systems to assess mechanisms and potential interventions.
Collapse
Affiliation(s)
- Tavé van Zyl
- Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear, Boston, MA 02114;
- Center for Brain Science, Harvard University, Cambridge, MA 02138
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
| | - Wenjun Yan
- Center for Brain Science, Harvard University, Cambridge, MA 02138
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
| | - Alexi McAdams
- Department of Ophthalmology, Harvard Medical School and Massachusetts Eye and Ear, Boston, MA 02114
- Center for Brain Science, Harvard University, Cambridge, MA 02138
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
| | - Yi-Rong Peng
- Center for Brain Science, Harvard University, Cambridge, MA 02138
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
| | - Karthik Shekhar
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02142
- Koch Institute of Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142
| | - Aviv Regev
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02142
- Koch Institute of Integrative Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142
- Klarman Cell Observatory, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02142
| | - Dejan Juric
- Department of Medicine, Harvard Medical School and Massachusetts General Hospital Cancer Center, Boston, MA 02114
| | - Joshua R Sanes
- Center for Brain Science, Harvard University, Cambridge, MA 02138;
- Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138
| |
Collapse
|
|
27
|
Zhou Y, Xia X, Yang E, Wang Y, Marra KG, Ethier CR, Schuman JS, Du Y. Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential. FASEB J 2020; 34:7160-7177. [PMID: 32259357 PMCID: PMC7254553 DOI: 10.1096/fj.201902326r] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2019] [Revised: 02/24/2020] [Accepted: 03/23/2020] [Indexed: 12/13/2022]
Abstract
The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma.
Collapse
Affiliation(s)
- Yi Zhou
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan, China 410008
- Co-first author
| | - Xiaobo Xia
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan, China 410008
- Co-first author
| | - Enzhi Yang
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213
| | - Yiwen Wang
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, Hunan, China 410008
| | - Kacey G. Marra
- Departments of Plastic Surgery and Bioengineering, University of Pittsburgh, Pittsburgh, PA 15213
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| | - C. Ross Ethier
- Department of Biomedical Engineering, Georgia Institute of Technology & Emory University, Atlanta, GA 30332
| | - Joel S. Schuman
- Department of Ophthalmology, New York University School of Medicine, New York, NY 10016
| | - Yiqin Du
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213
- Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15213
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15213
| |
Collapse
|
|
28
|
Xiong S, Xu Y, Wang Y, Kumar A, Peters DM, Du Y. α5β1 Integrin Promotes Anchoring and Integration of Transplanted Stem Cells to the Trabecular Meshwork in the Eye for Regeneration. Stem Cells Dev 2020; 29:290-300. [PMID: 31854234 DOI: 10.1089/scd.2019.0254] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Stem cell-based therapy to restore the function of abnormal trabecular meshwork (TM) and decrease intraocular pressure (IOP) provides a novel approach to treat open-angle glaucoma. However, molecular mechanism for stem cells homing and anchoring to the TM remains unclear. This study aimed to discover the function of integrins in homing and integration of exogenous TM stem cells (TMSCs) to the TM. Integrin expression in TMSCs and fibroblasts was evaluated by quantitative real-time PCR (qPCR), flow cytometry, immunofluorescent staining, and western blotting. Expression of integrin ligand fibronectin was detected in cultured TM cells and murine TM tissue by immunostaining. Cell affinity to TM cells or fibronectin matrix was examined to compare TMSCs with TMSCs functionally blocked with an α5β1 integrin antibody. TMSCs and TMSCs with α5β1 integrin-blocking were intracamerally injected into wild-type mice. Wholemounts and cryosections were analyzed to discover cell distribution and integration at 3 days and 1 month. IOP was measured to detect possible changes. We discovered that human TMSCs expressed a higher level of α5β1 integrin than fibroblasts, but similar levels of αvβ3 and αvβ5 integrin. Upregulation of fibronectin was found in both TM cells treated with dexamethasone for 14 days and murine TM tissues damaged by laser photocoagulation. TMSCs were able to attach to the TM cells and fibronectin matrix in vitro. When the surface α5β1 integrin was blocked, the attached cell numbers were significantly reduced. Both TMSCs and TMSCs incubated with an α5β1 integrin-blocking antibody could home to the mouse TM after injection. TMSCs blocked with the α5β1 integrin-blocking antibody were not retained in the TM tissue at 1 month. The injected cells did not affect mouse IOP. In conclusion, highly expressed α5β1 integrin participates in maintaining TMSCs anchored and integrated to the TM, which would be crucial for stem cell-based therapy for glaucoma.
Collapse
Affiliation(s)
- Siqi Xiong
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China.,Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Yi Xu
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Yiwen Wang
- Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China.,Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Ajay Kumar
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Donna M Peters
- Department of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin
| | - Yiqin Du
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania.,Department of Developmental Biology, University of Pittsburgh, Pittsburgh, Pennsylvania.,McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| |
Collapse
|
|
29
|
Yemanyi F, Vranka J, Raghunathan V. Generating cell-derived matrices from human trabecular meshwork cell cultures for mechanistic studies. Methods Cell Biol 2020; 156:271-307. [PMID: 32222223 DOI: 10.1016/bs.mcb.2019.10.008] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Ocular hypertension has been attributed to increased resistance to aqueous outflow often as a result of changes in trabecular meshwork (TM) extracellular matrix (ECM) using in vivo animal models (for example, by genetic manipulation) and ex vivo anterior segment perfusion organ cultures. These are, however, complex and difficult in dissecting molecular mechanisms and interactions. In vitro approaches to mimic the underlying substrate exist by manipulating either ECM topography, mechanics, or chemistry. These models best investigate the role of individual ECM protein(s) and/or substrate property, and thus do not recapitulate the multifactorial extracellular microenvironment; hence, mitigating its physiological relevance for mechanistic studies. Cell-derived matrices (CDMs), however, are capable of presenting a 3D-microenvironment rich in topography, chemistry, and whose mechanics can be tuned to better represent the network of native ECM constituents in vivo. Critically, the composition of CDMs may also be fine-tuned by addition of small molecules or relevant bioactive factors to mimic homeostasis or pathology. Here, we first provide a streamlined protocol for generating CDMs from TM cell cultures from normal or glaucomatous donor tissues. Second, we document how TM cells can be pharmacologically manipulated to obtain glucocorticoid-induced CDMs and how generated pristine CDMs can be manipulated with reagents like genipin. Finally, we summarize how CDMs may be used in mechanistic studies and discuss their probable application in future TM regenerative studies.
Collapse
Affiliation(s)
- Felix Yemanyi
- Department of Basic Sciences, University of Houston, Houston, TX, United States
| | - Janice Vranka
- Casey Eye Institute, Oregon Health and Science University, Portland, OR, United States
| | - VijayKrishna Raghunathan
- Department of Basic Sciences, University of Houston, Houston, TX, United States; The Ocular Surface Institute, College of Optometry, University of Houston, Houston, TX, United States; Department of Biomedical Engineering, Cullen College of Engineering, University of Houston, Houston, TX, United States.
| |
Collapse
|
|
30
|
Stem cells from trabecular meshwork cells can secrete extracellular matrix. Biochem Biophys Res Commun 2020; 523:522-526. [PMID: 31902587 DOI: 10.1016/j.bbrc.2019.12.080] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2019] [Accepted: 12/17/2019] [Indexed: 01/01/2023]
Abstract
Isolation of trabecular meshwork stem cells in vitro provides the foundation of a novel treatment for glaucoma. Trabecular meshwork stem cells (TMSCs) of the fetal calve were extracted and cultured for this experiment. TMSCs were isolated through side population cell sorting. TMSCs were then identified using immunofluorescent staining. Extracellular matrix (ECM) expression in TM cells derived from TMSCs was evaluated with Western blot. Our results showed a positive expression of stem cell markers Notch1 and OCT-3/4 in TMSCs, but no TM cells markers TIMP3 or AQP1. In contrast, primary TM cells expressed these TM cell markers but no stem cell markers. Our result confirmed that there are expression of ECM components, such as fibronectin, laminin, collagen I and collagen IV in TM cells differentiated from TMSCs. CONCLUSION: TM cells derived from TMSCs can secrete ECM components which is important for sustain the physiological function.
Collapse
|
|
31
|
Kumar A, Xu Y, Du Y. Stem Cells from Human Trabecular Meshwork Hold the Potential to Develop into Ocular and Non-Ocular Lineages After Long-Term Storage. Stem Cells Dev 2020; 29:49-61. [PMID: 31680626 PMCID: PMC6931915 DOI: 10.1089/scd.2019.0169] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2019] [Accepted: 11/01/2019] [Indexed: 01/10/2023] Open
Abstract
Stem cells from the eye hold a great potential for vision restoration and can also be used for regeneration in other tissues. In this study, we characterized the stem cell properties of Trabecular meshwork stem cells (TMSCs) after long-term cryopreservation (∼8 years). TMSCs derived from four donors were examined for their viability and proliferation, as well as stem cell marker expression. Spheroid formation, colony formation, and multipotency were investigated. We observed that TMSCs were fully viable with variable proliferation ability. They expressed the stem cell markers CD90, CD166, CD105, CD73, OCT4, SSEA4, Notch1, KLF4, ABCG2, Nestin, and HNK1 detected by flow cytometry, quantitative polymerase chain reaction, or immunofluorescent staining. They could form spheroids and colonies after thawing. All TMSCs were able to differentiate into osteocytes, neural cells, and trabecular meshwork (TM) cells, but not adipocytes. Differentiated TM cells responded to dexamethasone treatment with increased expression of myocilin and angiopoietin-like 7 (ANGPTL7). In a nutshell, our study demonstrated that TMSCs retain their stem cell properties after long-term cryopreservation and hence can be an effective cell therapy source for various clinical applications.
Collapse
Affiliation(s)
- Ajay Kumar
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Yi Xu
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
| | - Yiqin Du
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Pennsylvania
- Department of Developmental Biology, University of Pittsburgh, Pittsburgh, Pennsylvania
- McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania
| |
Collapse
|
|
32
|
Zhang J, Wang NL. Progression on canaloplasty for primary open angle glaucoma. Int J Ophthalmol 2019; 12:1629-1633. [PMID: 31637200 DOI: 10.18240/ijo.2019.10.16] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2019] [Accepted: 04/01/2019] [Indexed: 12/20/2022] Open
Abstract
As a non-penetrating glaucoma surgery (NPGS), canaloplasty aims to reconstruct the physiological outflow of aqueous humor by dilating the Schlemm's canal. Ab interno canaloplasty (ABiC), which can reconstruct the natural outflow pathways of aqueous humor in mild-to-moderate primary open angle glaucoma (POAG) patients, is a new minimally invasive glaucoma surgery (MIGS) procedure improving from traditional canaloplasty. Canaloplasty can reduce intraocular pressure (IOP) with high efficiency and security. There are no complications such as scar formation and encapsulation for this no-bleb canaloplasty.
Collapse
Affiliation(s)
- Jun Zhang
- Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing 100005, China.,Dalian Aier Eye Hospital, Dalian 116031, Liaoning Province, China
| | - Ning-Li Wang
- Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing 100005, China
| |
Collapse
|
|
33
|
Castro A, Du Y. Trabecular Meshwork Regeneration - A Potential Treatment for Glaucoma. CURRENT OPHTHALMOLOGY REPORTS 2019; 7:80-88. [PMID: 31316866 DOI: 10.1007/s40135-019-00203-2] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Purpose In this review, we overview the pathophysiology of primary open-angle glaucoma as it relates to the trabecular meshwork (TM), exploring modes of TM dysfunction and regeneration via stem cell therapies. Recent Findings Stem cells from a variety of sources, including trabecular meshwork, mesenchymal, adipose and induced pluripotent stem cells, have shown the potential to differentiate into TM cells in vitro or in vivo and to regenerate the TM in vivo, lowering intraocular pressure (IOP) and reducing glaucomatous retinal ganglion cell damage. Summary Stem cell therapies for TM regeneration provide a robust and promising suite of treatments for eventual lowering of IOP and prevention of glaucomatous vision loss in humans in the future. Further investigation into stem cell homing mechanisms and the safety of introducing these cells into human anterior chamber, for instance, are required before clinical applications in treating glaucoma patients.
Collapse
Affiliation(s)
- Alexander Castro
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213.,University of Virginia, Charlottesville, VA 22904
| | - Yiqin Du
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PA 15213.,McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15213.,Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15213
| |
Collapse
|
|
34
|
Adams CM, Stacy R, Rangaswamy N, Bigelow C, Grosskreutz CL, Prasanna G. Glaucoma - Next Generation Therapeutics: Impossible to Possible. Pharm Res 2018; 36:25. [PMID: 30547244 DOI: 10.1007/s11095-018-2557-4] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2018] [Accepted: 11/12/2018] [Indexed: 12/28/2022]
Abstract
The future of next generation therapeutics for glaucoma is strong. The recent approval of two novel intraocular pressure (IOP)-lowering drugs with distinct mechanisms of action is the first in over 20 years. However, these are still being administered as topical drops. Efforts are underway to increase patient compliance and greater therapeutic benefits with the development of sustained delivery technologies. Furthermore, innovations from biologics- and gene therapy-based therapeutics are being developed in the context of disease modification, which are expected to lead to more permanent therapies for patients. Neuroprotection, including the preservation of retinal ganglion cells (RGCs) and optic nerve is another area that is actively being explored for therapeutic options. With improvements in imaging technologies and determination of new surrogate clinical endpoints, the therapeutic potential for translation of neuroprotectants is coming close to clinical realization. This review summarizes the aforementioned topics and other related aspects.
Collapse
Affiliation(s)
- Christopher M Adams
- Global Discovery Chemistry, Novartis Institutes for Biomedical Research (NIBR),, Cambridge, Massachusetts, USA
| | - Rebecca Stacy
- Translational Medicine, Ophthalmology, NIBR, Cambridge, Massachusetts, USA
| | - Nalini Rangaswamy
- Ophthalmology Research, Novartis Institutes for Biomedical Research, 22 Windsor Street, Cambridge, Massachusetts, 02139, USA
| | - Chad Bigelow
- Ophthalmology Research, Novartis Institutes for Biomedical Research, 22 Windsor Street, Cambridge, Massachusetts, 02139, USA
| | - Cynthia L Grosskreutz
- Ophthalmology Research, Novartis Institutes for Biomedical Research, 22 Windsor Street, Cambridge, Massachusetts, 02139, USA
| | - Ganesh Prasanna
- Ophthalmology Research, Novartis Institutes for Biomedical Research, 22 Windsor Street, Cambridge, Massachusetts, 02139, USA.
| |
Collapse
|
|
35
|
Human stem cells home to and repair laser-damaged trabecular meshwork in a mouse model. Commun Biol 2018; 1:216. [PMID: 30534608 PMCID: PMC6283842 DOI: 10.1038/s42003-018-0227-z] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2018] [Accepted: 11/14/2018] [Indexed: 12/13/2022] Open
Abstract
Glaucoma is the leading cause of irreversible vision loss, and reducing elevated intraocular pressure is currently the only effective clinical treatment. The trabecular meshwork is the main resistance site for aqueous outflow that maintains intraocular pressure. In this study, we transplanted human trabecular meshwork stem cells (TMSCs) intracamerally into mice that received laser photocoagulation over a 180° arc of the trabecular meshwork. TMSCs preferentially homed and integrated to the laser-damaged trabecular meshwork region and expressed differentiated cell markers at 2 and 4 weeks. Laser-induced inflammatory and fibrotic responses were prevented by TMSC transplantation with simultaneous ultrastructure and function restoration. Cell affinity and migration assays and elevated expression of CXCR4 and SDF1 in laser-treated mouse trabecular meshwork suggest that the CXCR4/SDF1 chemokine axis plays an important role in TMSC homing. Our results suggest that TMSCs may be a viable candidate for trabecular meshwork refunctionalization as a novel treatment for glaucoma. Hongmin Yun et al. show that implanted human stem cells can accurately home to and repair damaged trabecular meshwork tissue in the mouse eye via a chemokine axis defined by CXCR4 and SDF1. The study suggests that stem cells from the trabecular meshwork could be used to refunctionalize the outflow pathway as a treatment for glaucoma.
Collapse
|
|
36
|
Rabesandratana O, Goureau O, Orieux G. Pluripotent Stem Cell-Based Approaches to Explore and Treat Optic Neuropathies. Front Neurosci 2018; 12:651. [PMID: 30294255 PMCID: PMC6158340 DOI: 10.3389/fnins.2018.00651] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Accepted: 08/30/2018] [Indexed: 12/15/2022] Open
Abstract
Sight is a major sense for human and visual impairment profoundly affects quality of life, especially retinal degenerative diseases which are the leading cause of irreversible blindness worldwide. As for other neurodegenerative disorders, almost all retinal dystrophies are characterized by the specific loss of one or two cell types, such as retinal ganglion cells, photoreceptor cells, or retinal pigmented epithelial cells. This feature is a critical point when dealing with cell replacement strategies considering that the preservation of other cell types and retinal circuitry is a prerequisite. Retinal ganglion cells are particularly vulnerable to degenerative process and glaucoma, the most common optic neuropathy, is a frequent retinal dystrophy. Cell replacement has been proposed as a potential approach to take on the challenge of visual restoration, but its application to optic neuropathies is particularly challenging. Many obstacles need to be overcome before any clinical application. Beyond their survival and differentiation, engrafted cells have to reconnect with both upstream synaptic retinal cell partners and specific targets in the brain. To date, reconnection of retinal ganglion cells with distal central targets appears unrealistic since central nervous system is refractory to regenerative processes. Significant progress on the understanding of molecular mechanisms that prevent central nervous system regeneration offer hope to overcome this obstacle in the future. At the same time, emergence of reprogramming of human somatic cells into pluripotent stem cells has facilitated both the generation of new source of cells with therapeutic potential and the development of innovative methods for the generation of transplantable cells. In this review, we discuss the feasibility of stem cell-based strategies applied to retinal ganglion cells and optic nerve impairment. We present the different strategies for the generation, characterization and the delivery of transplantable retinal ganglion cells derived from pluripotent stem cells. The relevance of pluripotent stem cell-derived retinal organoid and retinal ganglion cells for disease modeling or drug screening will be also introduced in the context of optic neuropathies.
Collapse
Affiliation(s)
| | - Olivier Goureau
- Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France
| | - Gaël Orieux
- Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France
| |
Collapse
|
|
37
|
Stern JH, Tian Y, Funderburgh J, Pellegrini G, Zhang K, Goldberg JL, Ali RR, Young M, Xie Y, Temple S. Regenerating Eye Tissues to Preserve and Restore Vision. Cell Stem Cell 2018; 22:834-849. [PMID: 29859174 PMCID: PMC6492284 DOI: 10.1016/j.stem.2018.05.013] [Citation(s) in RCA: 99] [Impact Index Per Article: 14.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Ocular regenerative therapies are on track to revolutionize treatment of numerous blinding disorders, including corneal disease, cataract, glaucoma, retinitis pigmentosa, and age-related macular degeneration. A variety of transplantable products, delivered as cell suspensions or as preformed 3D structures combining cells and natural or artificial substrates, are in the pipeline. Here we review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing. While recognizing the challenges, we look forward to a deep portfolio of sight-restoring, stem cell-based medicine. VIDEO ABSTRACT.
Collapse
Affiliation(s)
- Jeffrey H Stern
- Neural Stem Cell Institute, Rensselaer, NY 12144, USA; Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA
| | - Yangzi Tian
- Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA
| | - James Funderburgh
- Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
| | - Graziella Pellegrini
- Centre for Regenerative Medicine, University of Modena and Reggio Emilia, via G.Gottardi 100, 41125 Modena, Italy
| | - Kang Zhang
- Shiley Eye Institute and Institute for Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093, USA; State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University and Guangzhou Regenerative Medicine and Health Laboratory, Guangzhou 510060, China
| | - Jeffrey L Goldberg
- Byers Eye Institute at Stanford University, 2452 Watson Court, Palo Alto, CA 94303, USA
| | - Robin R Ali
- Department of Genetics, University College London Institute of Ophthalmology, 11-43 Bath Street, London EC1V 9EL, UK; NIHR Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, City Road, London EC1V 2PD, UK; Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA
| | - Michael Young
- The Schepens Eye Research Institute, Massachusetts Eye and Ear, an affiliate of Harvard Medical School, Boston, MA 02114, USA
| | - Yubing Xie
- Colleges of Nanoscale Science and Engineering, SUNY Polytechnic Institute, 257 Fuller Road, Albany, NY 12203, USA
| | - Sally Temple
- Neural Stem Cell Institute, Rensselaer, NY 12144, USA; Kellogg Eye Center, University of Michigan, Ann Arbor, MI 48105, USA.
| |
Collapse
|
|
38
|
Dang Y, Waxman S, Wang C, Jensen A, Loewen RT, Bilonick RA, Loewen NA. Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model. PeerJ 2017; 5:e3629. [PMID: 28828244 PMCID: PMC5560227 DOI: 10.7717/peerj.3629] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2017] [Accepted: 07/10/2017] [Indexed: 12/13/2022] Open
Abstract
Objective The trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP) was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F) ablation (−80 °C × 2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI) assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64). IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to many PI-labeled, dead TM cells and calcein-labeled viable TM cells in S. Conclusion We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM extracellular matrix in an organotypic perfusion culture.
Collapse
Affiliation(s)
- Yalong Dang
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PAUnited States of America
| | - Susannah Waxman
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PAUnited States of America
| | - Chao Wang
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PAUnited States of America.,Department of Ophthalmology, Xiangya Hospital, Central South University, Changsha, China
| | - Adrianna Jensen
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PAUnited States of America
| | - Ralitsa T Loewen
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PAUnited States of America
| | - Richard A Bilonick
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PAUnited States of America
| | - Nils A Loewen
- Department of Ophthalmology, University of Pittsburgh, Pittsburgh, PAUnited States of America
| |
Collapse
|