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Molecular characterization of the anagen human hair follicle [Elektronische Ressource] / vorgelegt von Angela Ariza de Schellenberger

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221 pages
MOLECULAR CHARACTERIZATION OF THE ANAGENHUMAN HAIR FOLLICLEVorgelegt vonMasters of scienceAngela Ariza de Schellenbergeraus KolumbienVon der Fakultät III - ProzesswissenschaftenFachgebiet Medizinische BiotechnologieTechnische Universität Berlinzur Erlangung des akademischen GradesDoktor der Naturwissenschaften- Dr. rer. nat. -genehmigte DisssertationPromotionsausschuss:Vorsitzender: Prof. Dr. L.-A. GarbeBerichter: Prof. Dr. rer. nat. Roland Lauster Berrichter: Prof. Dr. med. Marcus MaurerTag der wissenschaftlichen Aussprache: 04. Juni 2010Berlin 2010D83“A theory is something nobody believes, except the person who made it.An experiment is something everybody believes, except the person who made it.”-Albert EinsteinIIABSTRACTHuman hair follicles are complete organs which develop early during embryogenesis and posses under normal conditions a long life self renewal capacity and skin repair po-tential under wounding conditions. The functioning of human hair follicles might be af-fected by several factors that are not yet fully understood.The full understanding of the ongoing mechanisms in hair follicle biology is of great relevance due to its impact on the research fields of dermatology, aging, cosmetics and tissue engineering.Much work has been done since the early sixties and extensive knowledge has been obtained about the potential of hair follicle cells.
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MOLECULAR CHARACTERIZATION OF THE ANAGEN
HUMAN HAIR FOLLICLE
Vorgelegt von
Masters of science
Angela Ariza de Schellenberger
aus Kolumbien
Von der Fakultät III - Prozesswissenschaften
Fachgebiet Medizinische Biotechnologie
Technische Universität Berlin
zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
- Dr. rer. nat. -
genehmigte Disssertation
Promotionsausschuss:
Vorsitzender: Prof. Dr. L.-A. Garbe
Berichter: Prof. Dr. rer. nat. Roland Lauster
Berrichter: Prof. Dr. med. Marcus Maurer
Tag der wissenschaftlichen Aussprache: 04. Juni 2010
Berlin 2010
D83“A theory is something nobody believes, except the person who made it.
An experiment is something everybody believes, except the person who made it.”
-Albert Einstein
IIABSTRACT
Human hair follicles are complete organs which develop early during embryogenesis
and posses under normal conditions a long life self renewal capacity and skin repair po-
tential under wounding conditions. The functioning of human hair follicles might be af-
fected by several factors that are not yet fully understood.
The full understanding of the ongoing mechanisms in hair follicle biology is of great
relevance due to its impact on the research fields of dermatology, aging, cosmetics and
tissue engineering.
Much work has been done since the early sixties and extensive knowledge has been
obtained about the potential of hair follicle cells. Nevertheless, the study of human hair
follicles is still limited due to the lack of specific markers that define stem cell popula-
tions and their progeny.
This work contributes to the characterization of epidermal, mesenchymal and extracellu-
lar matrix components of human hair follicles. Based on gene microarrays of laser mi-
crodissected anagen human hair follicles, it was possible to identify the in situ gene
expression of the epidermal and mesenchymal compartments, and compare the pro-
genitor cells with the correspondent transient amplifying cell populations.
On the basis of the in situ gene expression profiling was possible to revise expression of
known “markers”, and define new extracellular markers for epidermal and mesenchymal
stem cell niches and further differentiated cells populations at the mRNA level.
Using the same cell experimental design, the extracellular matrix components for grow-
ing (anagen) hair follicles were defined.
Relevant differences between connective tissue and dermal papilla fibroblasts, the two
mesenchymal components of hair follicles were described.
Cartilage oligomeric matrix protein (COMP) is detected in this work as an essential
component of the human hair follicle extracellular matrix through the hair cycle which is
specifically expressed by connective tissue fibroblasts in situ and in vitro.
In this work is hypothesized that altered COMP expression might be associated with
scant fine hair as in the case of some chondrodysplasia and scleroderma patients. To-
gether all this reveals for the first time that COMP is part of the ECM of human hair folli-
cles and suggests its important function in normal human hair biology.
IIIZusammenfassung
Menschliche Haarfollikel sind komplette Organe, die während der frühen Embryona-
lentwicklung gebildet werden und unter normalen Bedingungen lebenslang Fähigkeiten
zur Selbsterneuerung und zur Hautreparatur haben. Die Regulation der menschlichen
Haarfollikel wird durch einige Faktoren beeinflusst, deren Funktionen noch nicht kom-
plett bekannt sind. Die möglichst vollständige Aufklärung der Haarfollikel-Biologie ist,
aufgrund des großen Einflusses auf andere Wissenschaftsbereiche wie Dermatologie,
Alterung, Kosmetik und Tissue engineering, von großer Bedeutung.
Durch umfangreiche Forschungen seit den frühen Sechziger jahren hat sich ein um-
fangreiches Wissen über das Potential der Haarfollikel angesammelt. Trotzdem sind die
Untersuchungen über menschliche Haarfollikel, wegen des Fehlens spezifischer Marker
für Stammzellen und deren Abkömmlinge, immer noch limitiert.
Die vorliegende Arbeit trägt zur Charakterisierung von epidermalen, mesenchymalen
und extrazellulären Matrixkomponenten von menschlichen Haarfollikeln bei. Auf der Ba-
sis von Gen-Mikroarrays von Laser-Mikrodissezierten anagenen menschlichen Haarfol-
likeln war es möglich, die in situ-Genexpression von epidermalen und mesenchymalen
Kompartimenten zu messen und die Progenitorzellen mit den korrespondierenden, sich
transient-vermehrenden Zellpopulationen zu vergleichen.
Basierend auf der in situ-Genexpressionsprofilierung war es möglich, die Expression
von bekannten ,Markernʻ zu überprüfen, und neue extrazelluläre Marker für epidermale
und mesenchymale Stammzellnischen und für weiter differenzierte Zellpopulationen auf
dem mRNA-Level zu bestimmen.
Unter Nutzung der gleichen experimentellen Methoden konnten die Extrazellulärmatrix-
Komponenten für wachsende (anagene) Haarfollikel bestimmt werden.
Wichtige Unterschiede zwischen den Fibroblasten des Bindegewebes und des Papillar-
körpers, den beiden mesenchymalen Komponenten des Haarfollikels, konnten be-
schrieben werden.
Das oligomere Knorpel-Matrixprotein (cartilage oligomeric matrix protein – COMP) wur-
de in dieser Arbeit als essentielle Komponente der Extrazellulärmatrix des menschli-
chen Haarfollikels während des Haarzyklus gefunden, welches spezifisch durch die
Fibroblasten des Bindegewebe in situ und in vitro exprimiert wird.
In dieser Arbeit wird die Hypothese aufgestellt, dass geändert COMP Expression in
Verbindung mit spärlichem und dünnem Haar stehen kann, wie z.B. bei Chondrodyspla-
sie- und Sklerodermie-Patienten. Insgesamt wird zum ersten Mal gezeigt, dass COMP
ein Teil der ECM von humanen Haarfollikeln ist und eine wichtige Funktion in der huma-
nen Haarbiologie spielt.
IV" Abstract" " " " " " " " " " III
"Conte Index"""""""""V Supplmetal tables" " " " " " " " XII
" Abbreviations" " " " " " " " " XIII
1. Introduction! 1
1.1. Hair follicle morphology" 2
1.2. Human hair follicle morphogenesis" 4
1.3. Hair follicle cycle" 6
1.3.1. Regulators of hair follicle cycle" 9
1.4. Stem cell niches in human hair follicle" 10
1.5. Epithelial-mesenchymal interactions are essential in HF biology" 13
1.5.1. Wnt signaling pathway" 13
1.5.2. TGFβ signaling pathway" 15
1.5.3. p53 signaling pathway" 17
1.6. Epidermal originated cells (outer root sheath)" 18
1.6.1. Markers for ORS keratinocytes" 20
1.6.2. Keratins" 21
1.6.3. Quiescence" 22
1.7. Neuronal crest originated cells in the hair follicle" 22
1.7.1. Melanogenesis" 23
1.7.2. Markers for HF melanocytes" 25
1.8. Mesenchymal cells" 26
1.8.1. Markers for Dermal papilla cells" 28
1.8.2. Condensation capacity of HF mesenchymal cells" 29
1.8.3. Mesenchymal stromal cells characterization" 30
1.9. Hair follicle associated immune cells" 31
1.10. Extracellular matrix definition and formation" 31
1.10.1. Collagen IV" 33
1.10.2. Laminin" 33
V1.10.3. Integrins" 36
1.10.4. Proteoglycans" 37
1.10.5. Fibronectin" 38
1.10.6. Tenascin" 38
1.11. Cartilage oligomeric matrix protein (COMP)" 41
1.12. Addressed questions" 43
2. Materials and methods! 44
2.1. Materials" 44
2.1.1. Tissue" 44
2.1.2. Cell culture material" 44
2.1.3. Additional reagents" 44
2.1.4. Enzymes" 44
2.1.5. Solutions" 45
2.1.6. Kits" 46
2.1.7. Primers" 46
2.1.8. Machines" 47
2.1.9. Software" 47
2.1.10. Disposable material" 47
2.2. Methods" 48
2.2.1. Human tissue sampling" 48
2.2.2. Culture of hair follicle cells" 48
2.2.3. Matrices for cell culture" 49
2.2.4. Cryosections" 49
2.2.5. Immunocharacterization" 50
2.2.6. Amplification methods (TSA)" 53
2.2.7. Flow cytometry" 53
2.2.8. ApopTag Fluorescein In situ Apoptosis detection" 54
2.2.9. Differentiation methods" 55
VI2.2.10. Standard PCR" 56
2.2.11. In situ hybridization" 57
2.2.12. Laser microdissection" 58
2.2.13. Microarray Technology" 60
2.2.14. Quantitive PCR" 61
2.2.15. Statistical analysis" 62
2.3. Experimental design" 63
3. Results! 64
3.1. Characterization of the permanent vs non permanent outer root sheath
(ORS) of anagen hair follicles" 64
3.1.1. Differentiation stage of ORS keratinocytes" 64
3.1.1.1. Keratin expression of the PR and NP ORS keratinocytes64"
3.1.1.2. In situ hybridization for Keratin 15" 66
3.1.1.3. Expression of keratin 5, 10 and 17 in anagen VI HF." 68
3.1.2. Quiescent state of ORS cells" 71
3.1.2.1. Ki67/Apoptosis" 71
3.1.2.2. Survivin expression" 72
3.1.2.3. P63 Expression" 73
3.1.2.4. Summary of keratins and quiescence markers (Immunolabel-
ing)" 74
3.1.3. Microarray analysis of laser microdissected ORS cells" 75
3.1.3.1. Gene expression profile of immunolabeled keratins" 76
3.1.3.2. Gene expression profile of Immunolabeled quiescence mark-
ers" 77
3.1.4. Characterization of vimentin expressing melanocytes along the
ORS" 78
3.1.4.1. C-kit expressing cells along the ORS" 80
3.1.4.2. P-Mel17 expressing melanocytes along the ORS" 81
3.1.4.3. TRP1 expressing melanocytes along the ORS" 82
VII3.1.4.4. Summary of vimentin expressing melanocytes in human ana-
gen HF (Immunolabeling)." 83
3.1.4.5. Gene expression profile of melanocyte markers" 84
3.1.5. Preferentially expressed genes at the PR and NP ORS cells85"
3.1.5.1. Functional categories of genes with higher expression at the
permanent outer root sheath keratinocytes (PR ORS)" 87
3.1.5.2. Functional categories of genes with higher expression at the
non permanent outer root sheath (NP ORS)" 87
3.1.5.3. Wnt pathway gene expression profile at the ORS" 88
3.1.5.4. P53 pathway gene expression profile at the ORS" 89
3.1.5.5. TGFβ/BMP pathway gene expression profile at the ORS90"
3.1.6. Potential markers for the bulge/permanent outer root sheath (PR
ORS) keratinocytes" 91
3.2. Characterization of mesenchymal cells of anagen HF" 94
3.2.1. Microarray analysis between the DP and CT fibroblasts" 94
3.2.1.1. Expression profile of keratins, quiescent markers and melano-
cytic related genes in DP and CT fibroblasts." 95
3.2.1.2. Functional categories of genes with preferential expression in
the Dermal papilla cells (DP) or connective tissue fibroblasts
(CTF)." 96
3.2.1.3. Functional categories of genes with higher expression in der-
mal papilla (DP)" 97
3.2.1.4. Functional categories of genes with higher expression in con-
nective tissue fibroblasts (CTF)" 97
3.2.1.5. Wnt pathway expression profile in DP compared to CT fibro-
blasts" 98
3.2.1.6. TGFβ/BMP pathway expression profile in DP compared to CT
fibroblasts" 99
3.2.2. Markers for DP and CT fibroblasts" 100
3.2.2.1. Extracellular matrix components as “markers” for CT and DP
fibroblasts of the human HF." 100
3.2.2.2. Potential markers for dermal papilla cells (DP)" 102
3.2.3. Potential markers for connective tissue fibroblasts (CTF)" 102
3.3. The extracellular matrix components of anagen hair follicle" 103
VIII3.3.1. Expression of common ECM components" 104
3.3.1.1. Col IV Expression" 104
3.3.1.2. Fibronectin and α-Integrin 5 expression" 105
3.3.1.3. Laminin and α-Integrin 6 expression" 106
3.3.1.4. Tenascin expression" 107
3.3.1.5. Summary ECM components (Immunolabeling):" 108
3.3.2. Microarray analysis of the ECM components" 109
3.3.2.1. Gene expression profile of labeled ECM and BM components"
109
3.3.2.2. Gene expression profile of additional ECM components in an-
agen HFs." 111
3.3.3. Cartilage oligomeric matrix protein (COMP) expression in human
hair follicles" 115
3.4. Characterization of in vitro expanded CT and DP fibroblasts" 119
3.4.1. Expression of “mesenchymal markers” in situ" 123
3.4.1.1. The in situ expression of “Mesenchymal markers” and their in
vitro gene regulation." 124
4. Discussion! 128
4.1. Characterization of the permanent vs non permanent outer root sheath cells
of anagen hair follicles" 128
4.1.1. Differentiation stage of outer root sheath keratinocytes (Keratins
expression)" 128
4.1.2. Quiescent state of outer root sheath (ORS) cells" 130
4.1.3. Vimentin expressing melanocytes at the ORS" 132
4.1.4. Differences in the microarray data between the PR and NP ORS
cells" 136
4.1.5. Potential markers for the PR ORS (bulge) keratinocytes" 140
4.2. Characterization of mesenchymal cells in anagen HFs" 141
4.2.1. Microarray analysis between DP and CT fibroblasts" 142
4.2.1.1. Wnt pathway expression profile in anagen DP compared to CT
fibroblasts." 142
IX4.2.1.2. TGF-β/BMP pathway in anagen DP compared to CT fibro-
blasts" 143
4.2.2. Markers for DP and CT fibroblasts" 144
4.2.2.1. Extracellular matrix components are not optimal “markers” to
differentiate mesenchymal cells of anagen human HF." 144
4.2.2.2. Potential markers for DP and CT fibroblasts" 146
4.3. The extracellular matrix components of anagen hair follicle" 147
4.3.1. Collagen IV" 148
4.3.2. Fibronectin 1 and α5 integrin (ITGA5)" 148
4.3.3. Laminin and α6 Integrin" 149
4.3.4. Tenascin" 150
4.3.5. Microarray analysis of additional ECM components in anagen
HFs." 151
4.3.6. Cartilage oligomeric matrix protein (COMP) in human hair follicles"
158
4.4. Characterization of in vitro expanded CT and DP fibroblasts" 160
4.4.1. Expression of “Mesenchymal markers” in situ and in vitro."161
4.5. Proposed models for the HF stem cell niches" 163
4.5.1. The bulge niche" 163
4.5.2. The dermal papilla (DP) niche" 165
5. Conclusions! 168
5.1. What are the differences between PR ORS and NP ORS from anagen VI
human hair follicles?" 168
5.2. Which other molecules could be suggested as markers for the keratinocytes
stem cell population (PR ORS)?" 169
5.3. What are the major differences or similarities between the CT and the DP fi-
broblasts in vivo?" 170
5.4. Which molecules could be suggested as markers for the DP and the CT fi-
broblasts" 170
5.5. Is the extracellular matrix different at the PR and NP region?" 170
5.6. How to define DP and CT fibroblasts after in vitro expansion?" 172
6. Perspectives! 173
X