Photoreceptors are highly specialized neurons within the eye and the key retinal cells sensing light. They are indispensable for our visual perception and loss of photoreceptors consequently leads to loss of vision, a sense that alone is responsible for more than 30% of the input to our brain. Vision impairment and blindness is a leading cause of disability in the industrialized world and is in many cases ultimately due to a degeneration of the photoreceptors, which cannot be halted or reversed. Retinal degenerative diseases encompass a heterogeneous group of etiologies, mainly caused by various mutations in a plethora of proteins involved in the visual process. Currently, several therapeutic options are being explored, with so far one gene therapy for a rare inherited blinding condition being clinically approved. However, the gene therapy approach requires not only the presence of remaining photoreceptors but the tailoring of the therapy to each individual mutation. An alternative, more generally applicable approach is to restore vision through photoreceptor replacement therapy. As such, research on mouse-to-mouse photoreceptor transplantations has been carried out for many years, though with mixed results. In the last decade, it has however also become possible to generate large quantities of human photoreceptors through retinal organoid technology, allowing to instead transplant human cells. While promising, this field is still in development and principal conditions for successful photoreceptor transplantation have yet to be defined. Here, human-to-mouse photoreceptor transplantations were performed and assessed with the aim to receive insights into retinal cell replacement technology with specific focus on photoreceptor maturation, polarization and functional integration. Using a cone-degeneration host line, large-scale incorporation of human photoreceptor grafts into the murine retina was shown for the first time. It was found that for human photoreceptors, the choice of developmental stage strongly affects incorporation and maturation capacity. Furthermore, the results demonstrate the necessity of adequate graft-host interaction for successful transplant maturation and function, suggesting that photoreceptor replacement strategies might benefit from transplantation in earlier rather than late stages of retinal degeneration. Taken together, this thesis lays important groundwork for the further development of human photoreceptor replacement strategies to treat retinal degenerative disease.:ACKNOWLEDGEMENTS I
ABSTRACT III
ZUSAMMENFASSUNG V
PUBLICATIONS VII
TABLE OF CONTENTS IX
LIST OF FIGURES XIII
LIST OF TABLES XIV
GENERAL ABBREVIATIONS XV
GENE AND PROTEIN ABBREVIATIONS XVII
1 INTRODUCTION 1
1.1 THE RETINA AND LIGHT PERCEPTION 1
1.1.1 General structure of the eye 1
1.1.2 General structure of the retina 1
1.1.3 General photoreceptor structure 3
1.1.4 Phototransduction 4
1.1.5 Signal transmission to the brain 6
1.1.6 Major differences between rods and cones 7
1.1.7 The role of Müller glia in photoreceptor support and light perception 9
1.2 RETINAL DEGENERATION DISEASES AND TREATMENT OPTIONS 11
1.2.1 Retinal degeneration diseases 11
1.2.2 Therapeutic approaches to treat retinal degeneration diseases 12
1.3 CELL REPLACEMENT AS TREATMENT APPROACH FOR RETINOPATHIES 14
1.3.1 Transplantations of rodent retinal tissue and cells 14
1.3.2 Transplantations of human retinal tissue and cells 17
1.4 AIM OF THIS THESIS 22
2 CHARACTERIZATION OF CRX-MCHERRY HUMAN RETINAL ORGANOIDS AS PHOTORECEPTOR CELL SOURCE 23
2.1 AIMS 23
2.2 CHARACTERIZATION OF CRX-MCHERRY REPORTER-EXPRESSING CELLS 23
2.2.1 Crx-mCherry expression overlaps with endogenous CRX expression and increases over time 23
2.2.2 Crx-mCherry organoids contain an outer and an inner nuclear layer 24
2.2.3 Crx-mCherry+ cells express early and mature rod and cone markers 25
2.2.4 Crx-mCherry+ cells do not express proliferation markers 27
2.3 ENRICHMENT AND CHARACTERIZATION OF CRX-MCHERRY+ DONOR CELLS 28
2.3.1 Enrichment of Crx-mCherry+ cells by FACS 28
2.3.2 Characterization of Crx-mCherry enriched cells by single cell sequencing 29
2.3.3 Characterization of D200 Crx-mCherry-enriched cells by immunocytochemistry 30
2.4 SUMMARY 31
3 TRANSPLANTATION OF HUMAN CRX-MCHERRY+ GRAFTS AGED D100, D200 AND D300 INTO CPFL1 MICE 33
3.1 AIMS 33
3.2 CRX-MCHERRY+ CELLS OF ALL AGES CAN BE TRANSPLANTED AND SURVIVE IN THE MURINE RETINA 33
3.2.1 Human grafts can be identified by RCVRN staining 34
3.2.2 D100 Crx-mCherry+ transplants are larger than D200 and D300 grafts 34
3.2.3 Graft volume increase over time is not due to in vivo proliferation 36
3.3 GRAFT MORPHOLOGY DIFFERS WITH DONOR AGES 37
3.3.1 Human grafts can adopt an intraretinal position 37
3.3.2 Graft positioning changes over time 37
3.3.3 Qualitative differences in graft morphology between donor ages 38
3.4 GRAFT MATURATION 41
3.4.1 D200 but not D100 or D300 grafts develop large quantities of inner segments 41
3.4.2 Inner segment development is associated with close proximity to the host retina 42
3.5 HUMAN IDENTITY OF INTRARETINAL GRAFTS 43
3.5.1 Intraretinal Crx-mCherry+ grafts are largely a result of true morphological incorporation 43
3.5.2 Rare indications of potential human-to-mouse material transfer 45
3.6 SUMMARY 47
4 IN DEPTH CHARACTERIZATION OF TRANSPLANTED D200 CRX-MCHERRY+ CELLS 49
4.1 AIMS 49
4.2 EARLY POST TRANSPLANTATION DYNAMICS IN GRAFT POSITIONING AND GRAFT-HOST INTERACTIONS 49
4.2.1 Intraretinal and proximal D200 grafts interact with the host retina while isolated and distal clusters show only little interaction 49
4.2.2 Incorporation of D200 grafts is first evident at 8 weeks post transplantation 50
4.2.3 Host Müller glia extend processes into the graft before host bipolar cells 51
4.2.4 MG staining in D200 grafts originates from host MG 51
4.3 INCORPORATING D200 GRAFTS POLARIZE AND FORM STRUCTURES OF MATURE PHOTORECEPTORS 53
4.3.1 Grafts and host form an outer limiting membrane (OLM)-like structure 53
4.3.2 Inner segment formation occurs where an OLM is formed 54
4.3.3 Incorporating grafts form outer segment-like structures 55
4.3.4 Incorporating grafts form synaptic structures 57
4.3.5 Transplanted Crx-mCherry+ cells become enriched for cones 58
4.3.6 Higher levels of mature photoreceptor markers in ex vivo compared to in vitro cones 60
4.4 INCORPORATION AND MATURATION CAPACITY DEPEND ON THE HOST ENVIRONMENT 63
4.4.1 Graft morphology and maturation in C57BL/6JRj recipients resembles that in Cpfl1 hosts 63
4.4.2 Graft morphology and maturation in highly degenerated rd1 and tgCR host lines differs strongly from the outcome in models with an ONL 63
4.5 SUMMARY 67
5 FUNCTIONAL ASSESSMENT OF TRANSPLANTED CRX-MCHERRY+ CELLS 69
5.1 AIMS 69
5.2 HIGH-LEVEL FUNCTION 69
5.2.1 Light-Dark Box 69
5.3 TISSUE-LEVEL FUNCTION 71
5.3.1 Multi-electrode array assessment of D200+26w grafts in Cpfl1 mice 71
5.3.2 Isolation of cone-mediated RGC response through photopic light stimulation and L-AP4 addition 71
5.3.3 Graft-containing retinal portions exhibit cone-mediated light responses 72
5.4 SUMMARY 74
6 DISCUSSION AND FUTURE PERSPECTIVES 75
6.1 HUMAN GRAFTS CAN MORPHOLOGICALLY INCORPORATE INTO THE MODERATELY DEGENERATED MOUSE RETINA 75
6.2 INTRARETINAL GRAFTS MOSTLY REPRESENT TRUE INCORPORATION EVENTS, NOT MATERIAL TRANSFER 76
6.3 GRAFT MATURATION DEPENDS ON GRAFT-HOST INTERACTION 77
6.4 ESTABLISHMENT OF GRAFT-HOST INTERACTION AND GRAFT INCORPORATION 78
6.5 D200 CRX-MCHERRY+ CELLS ARE THE PREFERABLE DONOR POPULATION COMPARED TO D100 AND D300 80
6.6 CONES SHOW PREFERENTIAL SURVIVAL POST GRAFTING 81
6.7 FUNCTIONAL ANALYSES OF TRANSPLANTED ANIMALS 82
6.8 FUTURE CLINICAL TRANSLATION 85
6.9 MAJOR CONTRIBUTION TO OTHER WORK 88
7 FINAL CONCLUSION 89
8 MATERIALS AND METHODS 91
8.1 STUDY APPROVAL 91
8.2 MATERIALS 91
8.2.1 Materials and Chemicals 91
8.2.2 Cell Line 92
8.2.3 Mouse Lines 92
8.2.4 Antibodies 93
8.3 METHODS 95
8.3.1 Cell culture 95
8.3.2 Transplantations 96
8.3.3 Functional analyses 98
8.3.4 Immunohistochemistry and Immunocytochemistry 100
8.3.5 Imaging and image processing 103
8.3.6 Statistics 106
8.3.7 Single cell sequencing 107
8.3.8 Bioinformatic analysis 108
9 BIBLIOGRAPHY 111
10 APPENDIX 128
10.1 APPENDIX 1: ERKLÄRUNGEN ZUR ERÖFFNUNG DES PROMOTIONSVERFAHRENS 128
10.2 APPENDIX 2: BESTÄTIGUNG ÜBER EINHALTUNG DER AKTUELLEN GESETZLICHEN VORGABEN 129
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:84748 |
| Date | 18 April 2023 |
| Creators | Tessmer, Karen |
| Contributors | Ader, Marius, Becker, Catherina, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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