Photoreceptor replacement by transplantation is proposed as a treatment for blindness. Transplantation of healthy photoreceptor precursor cells into diseased murine eyes leads to the presence of ...functional photoreceptors within host retinae that express an array of donor-specific proteins. The resulting improvement in visual function was understood to be due to donor cells integrating within host retinae. Here, however, we show that while integration occurs the majority of donor-reporter-labelled cells in the host arises as a result of material transfer between donor and host photoreceptors. Material transfer does not involve permanent donor-host nuclear or cell-cell fusion, or the uptake of free protein or nucleic acid from the extracellular environment. Instead, RNA and/or protein are exchanged between donor and host cells in vivo. These data require a re-evaluation of the mechanisms underlying rescue by photoreceptor transplantation and raise the possibility of material transfer as a strategy for the treatment of retinal disorders.
Mesenchymal Stromal Cells (MSCs) are the most frequently used cell type in clinical studies, with over 1000 recorded ongoing or completed clinical trials. MSCs’ immunomodulatory properties have been ...extensively demonstrated in preclinical and many clinical studies. However, efficacy in Phase III clinical trials has been inconsistent. This has been proposed to be due to variable quality control and processing as well as incomplete understanding of mechanisms of action. A significant need to define critical quality attributes has been identified.
In 2006, ISCT defined minimal cell surface markers for MSC identity, with the aim of providing common measurable attributes to harmonise quality control. MSCs should be positive for CD73, CD90 and CD105, and negative for CD45, CD34, CD14, CD11b, CD19 and HLA-DR. Although these markers are widely used, quality control strategies and release criteria remain heterogeneous. Without standardised procedures, it is not possible to compare study outcomes or evaluate robustness of a given attribute.
As a means to address this, we are developing the first WHO International Reference Reagent for MSC therapies, designed to control the measurement of ISCT-defined minimal MSC identity markers in flow cytometry assays. To produce it, we will expand, fix and freeze-dry cells expressing MSC markers. Our work to date has focused on feasibility, comparison of candidate cell types and process optimisation. Key attributes need to be demonstrated for this material to meet the requirements of a reference reagent. Cells must express ISCT-defined positive MSC markers and lack expression of negative markers. Cells must be readily expandable, with marker expression profiles conserved through expansion, processing and in storage. Consistency is also paramount. A crucial requirement for a cell-based reference reagent is the ability to be scaled to meet demand and provide replacement banks as required.
The use of our reference reagent will add confidence to the measurement of MSC identity, and enable analytical traceability across batches, sources, products, and manufacturing pipelines. Reference reagents are particularly useful for demonstrating batch comparability, which is the second most commonly raised major objection against market authorisation applications for advanced therapy medicinal products. Ultimately, this will help developers to meet regulatory requirements and shorten the time for innovative medicines to be available to patients.
Previous work by the group has determined that post-mitotic rod photoreceptor precursors, when transplanted into adult mouse retinae, possess the ability to migrate into the recipient retinal outer ...nuclear layer (ONL) where photoreceptors reside, assume mature rod morphologies and restore some visual function in models of blindness. This thesis focuses on the modes and kinetics of transplanted cell migration and comparison of migration and maturation of transplanted rods to the analogous processes during retinal development. To examine the kinetics of migration and maturation of transplanted rod precursors, recipient retinae were examined at a range of time-points post-surgery. Transplanted rods that had migrated into the ONL were counted and their maturation was analysed in terms of morphological differentiation, rod markers expression and maturation of the nuclear chromatin architecture. These events were compared to the analogous processes in normal retinal development. To further understand the migration ability of transplanted rod precursors, I investigated migration trends of the integration-competent cell population during development. A pulse-chase approach was used to gauge average changes in rod precursor position with time after cell cycle exit and the dynamics of migration were analysed through real-time imaging of explanted retinae. Both approaches indicated bidirectional trajectories of rod migration during development. A parallel focus has been testing of the hypothesis that transplanted rods use recipient Müller glial cells, which span the depth of the retina, as scaffolds along which to migrate. The spatial relationship between transplanted rods and recipient Müller cells was confirmed using nearest neighbour analysis. Through real-time in vitro imaging of rod-Müller cell co-cultures, rods could be observed to elongate and move in a directed manner along Müller cell surfaces.
Previous work by the group has determined that post-mitotic rod photoreceptor precursors, when transplanted into adult mouse retinae, possess the ability to migrate into the recipient retinal outer ...nuclear layer (ONL) where photoreceptors reside, assume mature rod morphologies and restore some visual function in models of blindness. This thesis focuses on the modes and kinetics of transplanted cell migration and comparison of migration and maturation of transplanted rods to the analogous processes during retinal development. To examine the kinetics of migration and maturation of transplanted rod precursors, recipient retinae were examined at a range of time-points post-surgery. Transplanted rods that had migrated into the ONL were counted and their maturation was analysed in terms of morphological differentiation, rod markers expression and maturation of the nuclear chromatin architecture. These events were compared to the analogous processes in normal retinal development. To further understand the migration ability of transplanted rod precursors, I investigated migration trends of the integration-competent cell population during development. A pulse-chase approach was used to gauge average changes in rod precursor position with time after cell cycle exit and the dynamics of migration were analysed through real-time imaging of explanted retinae. Both approaches indicated bidirectional trajectories of rod migration during development. A parallel focus has been testing of the hypothesis that transplanted rods use recipient Müller glial cells, which span the depth of the retina, as scaffolds along which to migrate. The spatial relationship between transplanted rods and recipient Müller cells was confirmed using nearest neighbour analysis. Through real-time in vitro imaging of rod-Müller cell co-cultures, rods could be observed to elongate and move in a directed manner along Müller cell surfaces.
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