Immunological properties of embryonic stem cell-derived cardiomyocytes:
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| Sprache: | English |
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2018
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| 245 | 1 | 0 | |a Immunological properties of embryonic stem cell-derived cardiomyocytes |c vorgelegt von Dipl. Biol. Birte Baudis aus Wolfenbüttel, Germany |
| 264 | 1 | |a Köln |c 2018 | |
| 300 | |a 207 Seiten |b Illustrationen, Diagramme |c 21 cm | ||
| 336 | |b txt |2 rdacontent | ||
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| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Universität Köln |d 2018 | ||
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| 999 | |a oai:aleph.bib-bvb.de:BVB01-030809303 | ||
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| _version_ | 1804179304900198400 |
|---|---|
| adam_text | TABLE OF CONTENTS
TABLE OF
CONTENTS...............................................................................................................
3
A
BBREVIATIONS......................................................................................................................
7
ABSTRACT................................................................................................................................10
ZUSAMM
ENFASSUNG...........................................................................................................11
1
INTRODUCTION..........................................................................................................12
1.1 PLURIPOTENT STEM CELLS
(PSCS).............................................................................12
1.2 PSC-DERIVED
CARDIOMYOCYTES...............................................................................
13
1.3 PSC-CMS IN REGENERATIVE MEDICINE
...................................................................
14
1.4 ALLOGRAFT
REJECTION..................................................................................................15
1.4.1 MAJOR HISTOCOMPATIBILITY (MHC)
ANTIGENS.............................................................16
1.4.2
MIH-ANTIGENS..........................................................................................................18
1.4.3 THE PATHWAYS OF ALLORECOGNITION
..........................................................................
19
1.4.4 ACTIVATION OF NAIVE CD4+ AND CD8+ T
CELLS......................................................... 20
1.4.5 THE HUMORAL
IMMUNITY.........................................................................................21
1.4.6 NATURAL KILLER (NK)
CELLS........................................................................................22
1.4.7 THE COMPLEMENT
SYSTEM......................................................................................23
1.5 IMMUNOLOGICAL PROPERTIES OF
PSC-CMS..............................................................23
1.5.1 EXPRESSION OF IMMUNE-RELEVANT MOLECULES BY PSC-CMS
....................................
24
1.5.2 IMMUNOLOGICAL PROPERTIES OF PSC-CMS IN VITRO
....................................................
24
1.5.3 IMMUNOLOGICAL PROPERTIES OF PSC-CMS IN VIVO
....................................................
25
2 A IM
S........................................................................................................................32
3 MATERIALS AND M
ETHODS.....................................................................................
33
3.1
MATERIALS..................................................................................................................33
3.1.1 CELL CULTURE
REAGENTS.............................................................................................33
3.1.2 MEDIUM
FORMULATIONS.............................................................................................35
3.1.3 MATERIALS FOR
TRANSPLANTATION.................................................................................
36
3.1.4 TRANSPLANTATION
EQUIPMENT...................................................................................
37
3.1.5
CONSUMABLES........................................................................................................
38
3.1.6 MOLECULAR BIOLOGY REAGENTS, KITS AND
MATERIALS...................................................40
3.1.7
BUFFERS...................................................................................................................43
3.1.6 KITS AND
PLASMIDS................................................................................................
44
3.1.9 LABORATORY
APPARATUS...........................................................................................44
3.1.10 LABORATORY
APPARATUS............................................................................................
45
3.1.11
SOFTWARE................................................................................................................
45
3.1.12 PCR PRIMERS USED FOR GENE EXPRESSION
ANALYSES...............................................46
3.1.13 LIVE/DEAD STAINING
DYES........................................................................................48
3.1.14 PRIMARY AND SECONDARY
ANTIBODIES.......................................................................48
3.1.15 CELL
LINES.................................................................................................................51
3.1.16 IPSC
LINE.................................................................................................................52
3.1.17 FIBROBLAST CELL
LINES...............................................................................................
52
3.1.18 MOUSE LYMPHOMA CELL LINES EL4 AND
E.G7-OVA................................................53
3.1.19 MELANOMA CELL LINES 816 AND 816-OVA
(MO4-10)..............................................53
3.1.20 B3Z HYBRIDOMA
CELLS.............................................................................................
54
3.1.21
ANIMALS...................................................................................................................54
3.2
METHODS...................................................................................................................56
3.2.1
COATING...................................................................................................................56
3.2.2 GENERATION OF
PSC-CMS.......................................................................................57
3.2.3 ENZYMATIC DISSOCIATION OF
PSC-CMS....................................................................58
3.2.4 FLOW CYTOMETRIC LIVE CELL
STAINING..........................................................................
58
3.2.5 RNA ISOLATION AND QUALITY
CONTROL.........................................................................60
3.2.6 CDNA
SYNTHESIS.....................................................................................................61
3.2.7 PCR
AMPLIFICATION..................................................................................................61
3.2.6 SEMI-QUANTITATIVE REAL TIME PCR
.........................................................................
62
3.2.9 B3Z HYBRIDOMA
ASSAY...........................................................................................
63
3.2.10 ANALYSIS OF CELL SURFACE AREA
................................................................................
64
3.2.11 ACTIVATION AND ISOLATION OF H-2KB-SIINFEKL-SPECIFIC CD8+ CTLS
.......................
64
3.2.12 ISOLATION OF CD8A+
CTLS........................................................................................64
3.2.13 CHROMIUM RELEASE
ASSAY......................................................................................65
3.2.14 INTERFERON-Y ENZYME-LINKED IMMUNOSORBENT ASSAY
(ELISA).................................66
3.2.15 SIINFEKL-H-2KB-SPECIFIC CD8+ CTL KILLING OF ESC-CMS MONITORED BY
TIME
LAPSE
MICROSCOPY...................................................................................................66
3.2.16 QUANTIFICATION OF ESC-CM LYSIS DURING LONG-TERM CTL
ASSAY...............................67
3.2.17 KIDNEY CAPSULE
TRANSPLANTATION............................................................................
67
3.2.18 BLOOD WITHDRAWAL AND SERUM ISOLATION
.................................................................
70
3.2.19 IMMUNOHISTOCHEMISTRY AND IMMUNOCYTOCHEMISTRY
.............................................
71
3.2.20 ANTIBODY
VALIDATION................................................................................................
72
3.2.21 QUANTIFICATION OF IMMUNE CELL INFILTRATION AND THE CASPASE-3
SIGNAL
.....................
73
3.2.22 HUMORAL
RESPONSE..................................................................................................74
3.2.23 OVA IMMUNIZATION BY TAIL VEIN
INJECTION................................................................75
3.2.24 DEXTRAMER* ASSAY - DETECTION OF ANTIGEN-SPECIFIC T-CELLS WITH MHC
MULTIMERS...............................................................................................................75
3.2.25 CYTOKINE ASSAY BY FLOW
CYTOMETRY........................................................................77
3.2.26 STATISTICAL
ANALYSIS.................................................................................................77
4
RESULTS....................................................................................................................79
4.1 CELL MODEL FOR STUDYING THE IMMUNOGENICITY OF ESC-CMS
.............................
79
4.1.1 VALIDATION OF OVA EXPRESSION AND SIINFEKL-H-2KB
PRESENTATION.....................81
4.1.2 EXPRESSION OF MHC CLASS II, CD80 AND CD86 MOLECULES ON ESC-CMS
.............
86
4.1.3 ESC-CMS BASICALLY DID NOT EXPRESS THE IMMUNOGENIC MIH-ANTIGENS
ZG-16
OR
HORMAD-1...........................................................................................................90
4.2 VALIDATION OF THE IN VIVO TRANSPLANTATION SYSTEM
.............................................
92
4.2.1 THE KIDNEY CAPSULE IS AN IMMUNOCOMPETENT SITE
...............................................
93
4.2.2 ESC-DERIVED CARDIAC CLUSTERS SURVIVE UNDER THE KIDNEY CAPSULE FOR
AT LEAST
3
MONTHS................................................................................................................97
4.2.3 THE MOST SIGNIFICANT LOSS OF CMS UNDER THE KIDNEY CAPSULE OF
RAG2 /YC*/
MICE OCCURS PERI- AND EARLY
POSTOPERATIVE.........................................................100
4.2.4 ASSESSMENT OF APOPTOSIS IN CARDIAC CLUSTERS PRIOR TO AND AFTER
TRANSPLANTATION.....................................................................................................102
4.3 MIH-MISMATCHED ESC-CMS WERE NOT REJECTED UPON TRANSPLANTATION
UNDER THE KIDNEY CAPSULE WITHIN 8
WEEKS........................................................ 106
4.4 ESC-CMS WERE RETAINED UNDER THE KIDNEY CAPSULES OF ALLOGENEIC MHC-
MISMATCHED BALB/C M
ICE.................................................................................
110
4.5 ESC-CMS WERE REJECTED UNDER THE KIDNEY CAPSULES OF MHC-MISMATCHED
FVB/N
MICE...........................................................................................................113
4.6 IMMUNE CELL
INFILTRATION.......................................................................................114
4.6.1 IMMUNE CELL INFILTRATION IN IMMUNODEFICIENT RAG2 /*YC /
MICE...............................115
4.6.2 IMMUNE CELL INFILTRATION IN MIH-MISMATCHED 129S2/SVPAS M ICE
......................
119
4.6.3 IMMUNE CELL INFILTRATION IN MHC-MISMATCHED BALB/C
MICE...............................119
4.6.4 IMMUNE CELL INFILTRATION IN MHC-MISMATCHED FVB/N
MICE.................................120
4.7 HUMORAL
RESPONSE...............................................................................................
121
4.8 SUSCEPTIBILITY OF APIG44-OVA-CMS TO PRE-ACTIVATED SIINFEKL-H-2KB-
SPECIFIC CD8+ CTLS IN VIVO
.................................................................................
125
4.8.2 ADJ+OVA-IMMUNIZATION INDUCED REJECTION OF OVA-EXPRESSING B16
MELANOMA CELLS IN MHC-MATCHED 129S2/SVPAS
MICE......................................129
4.8.3 INDUCTION OF SIINFEKL-H-2KB-SPECIFIC CD8+ CTLS BY IMMUNIZATION
WITH
ADJ+OVA DOES NOT AFFECT THE SURVIVAL OF TRANSPLANTED APIG44-OVA-CMS IN
MIH-MISMATCHED RECIPIENTS
................................................................................
131
4.8.4 RE-ACTIVATION OF H-2KB-SIINFEKL-SPECIFIC CD8+ CTLS BY A BOOSTER
DOSE OF
OVA ALSO DID NOT AFFECT THE SURVIVAL OF TRANSPLANTED APIG44-OVA-CMS IN
MHC-MATCHED GRAFT RECIPIENTS
...........................................................................
135
4.9 SUSCEPTIBILITY OF APIG44-OVA-CMS TOWARDS LYSIS BY ACTIVATED
SIINFEKL-H-2KB-SPECIFIC CD8+ CTLS IN
VITRO................................................... 140
4.10 CTL-MEDIATED KILLING OF APIG44-OVA-CMS BECAME EVIDENT ONLY AFTER
IFN-Y TREATMENT AND SIINFEKL-IOADING OF
CMS...............................................142
4.11 SIINFEKL-H-2KB-SPECIFIC CD8+ CTLS RECOGNIZE OVA-EXPRESSING CMS AS
DEMONSTRATED BY IFN-Y
SECRETION......................................................................
143
4.12 KILLING OF ADHERENT APIG44-OVA-CMS BY SIINFEKL-H-2KB-SPECIFIC CD8+
CTLS REVEALED BY OBSERVATION OVER LONGER PERIODS OF TIME USING TIME
LAPSE
MICROSCOPY.................................................................................................145
5 D
ISCUSSION.........................................................................................................
151
5.1 VALIDATION OF THE TRANSPLANTATION MODEL
..........................................................
152
5.1.1 LONG-TERM SURVIVAL OF ESC-CMS UNDER THE KIDNEY
CAPSULE...............................152
5.1.2 THE KIDNEY CAPSULE IS AN IMMUNOCOMPETENT TRANSPLANTATION SITE
...................
153
5.2 ESC-CMS WERE ACCEPTED UNDER THE KIDNEY CAPSULE OF MHC-MISMATCHED
BALB/C MICE, BUT REJECTED FROM FVB/N M
ICE..................................................154
5.2.1 REJECTION AND CELLULAR RESPONSE
.........................................................................
154
5.2.2 HUMORAL RESPONSE TO
ESC-CMS.........................................................................
159
5.3 IMMUNE RESPONSE TO MIH-MISMATCHED ESC-CMS
..........................................
164
5.4 IN VITRO SUSCEPTIBILITY OF ESC-CMS TOWARDS CTL LYSIS
...................................
170
5.5
SUMMARY................................................................................................................173
5.6
OUTLOOK..................................................................................................................174
6 SUPPLEMENTARY INFORM
ATION..........................................................................
176
6.1 ACTIVE CASPASE-3
STAINING..................................................................................176
6.2 OVERVIEW OF P-VALUES OF THE STATISTICAL ANALYSIS OF INFILTRATING
CD45
CD3+ AND F4/80+ CELLS
..........................................................................................
176
6.3 HUMORAL
RESPONSE................................................................................................178
6.4 SUSCEPTIBILITY OF APIG44-OVA-CMS TO PRE-ACTIVATED SIINFEKL-H-2KB-
SPECIFIC CDS* CTLS IN
VIVO..................................................................................182
6.5 SUSCEPTIBILITY OF APIG44-OVA-CMS TOWARDS LYSIS BY ACTIVATED
SIINFEKL-H-2KB-SPECIFIC CD8+ CTLS IN
VITRO...................................................183
7
CONTRIBUTIONS.....................................................................................................189
8 R
EFERENCES.........................................................................................................
190
FIGURE
INDEX.....................................................................................................................
200
TABLE
INDEX........................................................................................................................203
ACKNOWLEDGEMENTS.......................................................................................................
204
ERKLAERUNG...........................................................................................................................207
|
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| spelling | Baudis, Birte Verfasser (DE-588)115709922X aut Immunological properties of embryonic stem cell-derived cardiomyocytes vorgelegt von Dipl. Biol. Birte Baudis aus Wolfenbüttel, Germany Köln 2018 207 Seiten Illustrationen, Diagramme 21 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Köln 2018 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf http://d-nb.info/1162363185/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030809303&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Baudis, Birte Immunological properties of embryonic stem cell-derived cardiomyocytes |
| subject_GND | (DE-588)4113937-9 |
| title | Immunological properties of embryonic stem cell-derived cardiomyocytes |
| title_auth | Immunological properties of embryonic stem cell-derived cardiomyocytes |
| title_exact_search | Immunological properties of embryonic stem cell-derived cardiomyocytes |
| title_full | Immunological properties of embryonic stem cell-derived cardiomyocytes vorgelegt von Dipl. Biol. Birte Baudis aus Wolfenbüttel, Germany |
| title_fullStr | Immunological properties of embryonic stem cell-derived cardiomyocytes vorgelegt von Dipl. Biol. Birte Baudis aus Wolfenbüttel, Germany |
| title_full_unstemmed | Immunological properties of embryonic stem cell-derived cardiomyocytes vorgelegt von Dipl. Biol. Birte Baudis aus Wolfenbüttel, Germany |
| title_short | Immunological properties of embryonic stem cell-derived cardiomyocytes |
| title_sort | immunological properties of embryonic stem cell derived cardiomyocytes |
| topic_facet | Hochschulschrift |
| url | http://d-nb.info/1162363185/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=030809303&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT baudisbirte immunologicalpropertiesofembryonicstemcellderivedcardiomyocytes |
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