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Purification Of Progenitor Photoreceptors Derived From The Directed Differentiation Of Human Pluripotent Stem Cells

Weil, BD; (2017) Purification Of Progenitor Photoreceptors Derived From The Directed Differentiation Of Human Pluripotent Stem Cells. Doctoral thesis , UCL (University College London). Green open access

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Abstract

Cell therapy has the potential to treat a wide variety of unmet therapeutic indications that affect a growing number of people globally. Many of these therapies require purification steps to separate specific cell types from heterogeneous populations. This thesis investigates current affinity purification platforms to isolate human pluripotent stem cell-derived progenitor photoreceptors for the treatment of retinal dystrophies, and introduces a novel purification technology which possess bioprocessing and clinical advantages over current techniques. Successful production of progenitors was achieved using both induced pluripotent stem cells (iPSC) and human embryonic stem cells (hESC). By controlling the cell aggregation step and other iterative improvements to the retinal differentiation, 35.7% of cells generated expressed Cone-Rod Homeobox (CRX)-positive – a key marker to define progenitor photoreceptors. The critical performance metrics of fluorescent-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) were then derived through experimentation. Sort purity, progenitor yield and viable cell recovery of CD73-positive cell populations – a surface marker shown to co-express with CRX - were measured, and demonstrated that high purity separations above 90% were attained. However, both methods suffered from low cell recoveries with over 30 or 40% of cells (for FACS and MACS respectively) lost through the numerous processing steps involved in labelling cells with either a fluorescent or paramagnetic tag, washing and sorting samples. Cell labelling also leaves the product with a bound cellular label, complicating additional processing and potentially causing toxic clinical affects. A novel purification technology was assessed with SpheriTech affinity beads that possess bioprocess and clinical advantages over current purification methods. Cells are unmodified through isolation, with the positively selected cell type remaining label-free after processing. Consequently, cells experience minimal process steps so the time, risk, cost burden of purification and cell loss is reduced. Comparable purity with all separations was observed, however progenitor yield was noted to be lower with SpheriTech affinity beads than for FACS and MACS. To assess the impact different purification technologies have upon the complete bioprocess in an iPSC-derived therapy, an economic cost-modelling tool was created. By inputting experimentally-derived data into an integrated model, the cost of goods (COG) per dose was evaluated when using each of the three affinity purification methods. FACS was found to be economically favourable only at small production scales due to throughout limitations, with MACS presenting the most cost effective technology at all other scales. However, if progenitor yield could be increased to improve process yields through further process development, SpheriTech would compete with MACS across all scales tested.

Type: Thesis (Doctoral)
Title: Purification Of Progenitor Photoreceptors Derived From The Directed Differentiation Of Human Pluripotent Stem Cells
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
Keywords: Cell therapy, stem cell, retinal dystrophy, affinity purification, cell sorting, FACS, MACS
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Haematology
URI: https://discovery.ucl.ac.uk/id/eprint/1544170
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