'Validating the application of iPSC-derived Müller glia in a glaucoma therapy'

“验证 iPSC 来源的穆勒胶质细胞在青光眼治疗中的应用”

• 批准号: MR/P01660X/1
• 负责人: Gloria Limb
• 金额: $ 45.25万
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FP01660X%2F1
• 关键词: Validating; application; iPSC; derived; ller

Glaucoma is the commonest cause of irreversible blindness in the world. It is characterized by damage to specialized neurons of the retina (ganglion cells) and the optic nerve (the part that connects the eye to the brain). The research aims to validate the use of Müller glia derived from induced pluripotent stem cells (iPSC), for designing a cell therapy to treat advanced glaucoma. This proposal stems from our published work that showed that a population of cells from the adult human retina, known as 'Müller glia', have stem cell characteristics. When transplanted into the eye of animals with ganglion cell damage, these cells induced a significant recovery of visual function. We ascribed this function to the ability of these cells to physically and metabolically support the repair of ganglion cell axons, causing restoration of visual function. However, sourcing Müller glia from the adult human eye could limit the development of a therapy for use in a large population due to scarcity of tissue availability and the immune response that these cells can induce in a host. To facilitate the development of a cell therapy that could benefit many individuals, induced pluripotent stem cells (iPSC) may constitute a better cell choice as it might be possible to produce well characterized and traceable cells (ie, semi-defined cell preparations) for retinal therapies. In addition, development of iPSC banks (haplobanks) to derive iPSC compatible with many individuals, may facilitate cell transplantation in a large population cohort. On this basis, the main objectives of our proposed research are:1) To isolate and propagate Müller glia with stem cell characteristics from retinal organoids produced by iPSC in vitro, and to examine the genetic and physical properties of these cells, as well as their ability to produce nerve-protective factors in the laboratory: We will use iPSC and protocols provided by Prof Pete Coffey at the Institute of Ophthalmology, as well as protocols recently developed in our laboratory to isolate Müller cells from retinal organoids. These cells will be also examined for their ability to release factors that protect nerve cells. 2) To transplant iPSC-derived Müller cells into rat eyes with experimental glaucoma, and to assess visual function in these animals: To induce glaucoma, Brown Norway rats will be injected with magnetic beads into the anterior part of the eye to induce high intraocular pressure and consequent damage to the ganglion cells and the optic nerve (the main features of glaucoma). Animals will receive an intra-ocular injection of Müller cells (dead or alive) and their visual function will be assessed at various times (up to 24 weeks) after injection by measurement of their electrophysiological (ERG) response to light. We will also image in vivo the thickness of the nerve fibres that extend along the inner part of the retina towards the optic nerve. 3) To examine the ability of transplanted Müller cells to induce retinal ganglion cell/axon repair in the grafted eye using immuno-histochemical and electron-microscopy techniques, and to assess whether protective factors produced by Müller cells may be present in the transplanted retina: Histological and electron-microscopy examination of the transplanted retina will be undertaken at various times (up to 24 weeks) after transplantation to assess the integrity of host ganglion cells and to determine the presence of protective factors associated with the transplanted cells. We expect that the results will provide the basis for the future formulation of a pre-clinical development plan for the use of iPSC-derived Müller glia as a therapy for end stage glaucoma. This project will benefit from the availability of GMP iPSC lines derived by our collaborator Prof P. Coffey. Although iPSC have been derived from UK and US donors, Prof Coffey has proved the safety of UK derived stem cell lines in collaboration with the MRC PRION Unit.

青光眼是世界上最常见的导致不可逆性失明的原因。它的特点是视网膜(神经节细胞)和视神经(连接眼睛和大脑的部分)的专门神经元受到损害。这项研究旨在验证从诱导多能干细胞(IPSC)中提取的Müler胶质细胞用于设计治疗晚期青光眼的细胞疗法。这一建议源于我们发表的工作，该工作表明，来自成人视网膜的一组细胞，即所谓的Müler glia，具有干细胞特征。当将这些细胞移植到神经节细胞受损的动物的眼睛中时，这些细胞诱导了显著的视觉功能恢复。我们将这一功能归因于这些细胞在物理和代谢上支持神经节细胞轴突修复的能力，从而导致视觉功能的恢复。然而，由于组织可获得性和这些细胞可以在宿主中诱导的免疫反应的稀缺性，从成人眼睛中获取Müler胶质细胞可能会限制用于大规模人群的治疗方法的开发。为了促进细胞疗法的发展，使许多人受益，诱导多能干细胞(IPSC)可能是更好的细胞选择，因为它可能产生用于视网膜治疗的具有良好特性和可追踪的细胞(即，半定义细胞制剂)。此外，发展IPSC库(单库)以获得与许多个体相容的IPSC，可能会促进大规模人群中的细胞移植。在此基础上，我们提出的研究的主要目标是：1)体外从IPSC产生的视网膜器质中分离和培养具有干细胞特征的Müller胶质细胞，并检测这些细胞的遗传和物理性质，以及它们在实验室产生神经保护因子的能力：我们将使用IPSC和眼科研究所的Pete Coffey教授提供的方案，以及我们实验室最近开发的方案从视网膜器质中分离Müler细胞。还将检查这些细胞释放保护神经细胞的因子的能力。2)将IPSC来源的Müler细胞移植到实验性青光眼大鼠的眼内，并对这些动物的视功能进行评估：在诱导青光眼的过程中，Brown挪威大鼠将磁珠注射到眼球的前部，以诱导高眼压，继而对神经节细胞和视神经造成损害(青光眼的主要特征)。动物将接受眼内注射米勒细胞(死亡的或活的)，并将在注射后的不同时间(长达24周)通过测量它们对光的电生理(ERG)反应来评估它们的视觉功能。我们还将在体内成像沿着视网膜内部向视神经延伸的神经纤维的厚度。3)应用免疫组织化学和电子显微镜技术检测移植的Müler细胞诱导移植眼视网膜神经节细胞/轴突修复的能力，并评估移植视网膜中是否存在Müler细胞产生的保护因子：移植后不同时间(长达24周)将对移植的视网膜进行组织学和电子显微镜检查，以评估宿主神经节细胞的完整性，并确定与移植细胞相关的保护因素的存在。我们希望这些结果将为未来制定临床前开发计划提供基础，以使用IPSC来源的Müller胶质细胞作为终末期青光眼的治疗方法。该项目将受益于我们的合作者P.Coffey教授推出的GMP IPSC系列产品。尽管IPSC来自英国和美国的捐赠者，但科菲教授与MRC Prion单位合作，证明了英国来源的干细胞系是安全的。

项目成果

Strain Specific Responses in a Microbead Rat Model of Experimental Glaucoma.

• DOI: 10.1080/02713683.2020.1805472
• 发表时间: 2021-03
• 期刊: Current eye research
• 影响因子: 2
• 作者: Eastlake K;Jayaram H;Luis J;Hayes M;Khaw PT;Limb GA
• 通讯作者: Limb GA

Müller glia as an important source of cytokines and inflammatory factors present in the gliotic retina during proliferative vitreoretinopathy.

• DOI: 10.1002/glia.22942
• 发表时间: 2016-04
• 期刊: Glia
• 影响因子: 6.2
• 作者: Eastlake K;Banerjee PJ;Angbohang A;Charteris DG;Khaw PT;Limb GA
• 通讯作者: Limb GA

Short Communication: Contribution of Voltage-Gated Sodium Channels to the Rabbit Cone Electroretinograms.

短通讯：电压门控钠通道对兔锥体视网膜电图的贡献。

• DOI: 10.3109/02713683.2015.1029134
• 发表时间: 2016
• 期刊: Current eye research
• 影响因子: 2
• 作者: Becker S

Comparison of proteomic profiles in the zebrafish retina during experimental degeneration and regeneration.

• DOI: 10.1038/srep44601
• 发表时间: 2017-03-16
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Eastlake K;Heywood WE;Tracey-White D;Aquino E;Bliss E;Vasta GR;Mills K;Khaw PT;Moosajee M;Limb GA
• 通讯作者: Limb GA

Therapies for Retinal Degeneration - Targeting Common Processes

视网膜变性的治疗 - 针对常见过程

• DOI: 10.1039/9781788013666-00196
• 发表时间: 2018
• 作者: Eastlake K