Download Aromatic-turmerone induces neural stem cell proliferation in vitro

Aus dem Zentrum für Neurologie und Psychiatrie der Universität zu Köln
Klinik und Poliklinik für Neurologie
Direktor: Universitätsprofessor Dr. med. G. R. Fink
Aromatic-turmerone induces neural stem cell proliferation
in vitro and in vivo and promotes differentiation into neurons
Inaugural-Dissertation zur Erlangung der Würde
eines doctor rerum medicinalium
der Hohen Medizinischen Fakultät
der Universität zu Köln
vorgelegt von
Jörg Hucklenbroich
aus Wermelskirchen
promoviert am: 07. Oktober 2015
Gedruckt mit Genehmigung der Medizinischen Fakultät der Universität zu Köln (2015)
Dekan:
Universitätsprofessor Dr.med. Dr. h.c. Th. Krieg
1. Berichterstatterin:
Privatdozentin Dr. med. M. A. Rüger
2. Berichterstatter:
Professor Dr. rer. nat. A. Sachinidis
Erklärung
Ich erkläre hiermit, dass ich die vorliegende Dissertationsschrift ohne unzulässige Hilfe
Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die
aus fremden Quellen direkt oder indirekt übernommene Gedanken sind als solche kenntlich
gemacht.
Bei der Auswahl und der Auswertung des Materials, sowie bei der Herstellung des
Manuskriptes habe ich Unterstützungsleistungen von folgenden Personen erhalten:
Frau Privatdozentin Dr. med. M. Adele Rüger
Frau Silke Kirchen, M. A. Sc.
Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht beteiligt.
Insbesondere habe ich nicht die Hilfe einer Promotionsberaterin / eines Promotionsberaters
in Anspruch genommen. Dritte haben von mir weder unmittelbar noch mittelbar geldwerte
Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der vorgelegten
Dissertationsschrift stehen.
Die Dissertationsschrift wurde von mir bisher weder im Inland noch im Ausland in gleicher
oder ähnlicher Form einer anderen Prüfungsbehörde vorgelegt.
Köln, den 18. Dezember 2014
__________________
Jörg Hucklenbroich
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Die dieser Arbeit zugrunde liegenden Daten und Messergebnisse wurden von mir in
der
AG
„Neurale
Stammzellen“
mit
Unterstützung
und
Einführung
durch
Privatdozentin Dr. med. M. Adele Rüger und Prof. Dr. med. M. Schroeter ermittelt.
Die dem
in vivo Teil der Publikation zugrunde liegenden Daten wurden mit
Unterstützung von Privatdozentin Dr. med. M. Adele Rüger und Dr. med. Rebecca
Klein erhoben.
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Acknowledgements
Especially I want to thank my doctoral advisor, Dr. M. Adele Rüger, who supported
me during my entire thesis work with words and deeds. She was always available to
address my questions and problems and gave me valuable information and insights
and taught me the basics of immunology. Dr. Adele has an open mind and regarded
my ideas with interest, enabling many new approaches and results. With her kind
and friendly nature, she created an exemplary working climate and influenced our
working group to become a kind of family.
I especially want to thank Professor M. Schroeter for accepting me although I was a
senior doctoral candidate and for encouraging and supporting me, thus, helping me
develop as a scientist. Additionally, I am grateful for his special support in the
Osteopontin project, which has not been published yet. He taught me the basics,
making this work possible.
I also want to thank Dr. Rebecca Klein for introducing me to immunological protocols
and for her special support in generating the PET data for publication.
Additionally I am grateful to Anton Pikhovych, Ilja Bobylev, Claudia Drapatz and our
other colleagues in the “Neural Stem Cells “ and “Diseases of the Peripheral Nerve
System” groups for their support with experiments and many interesting talks and
ideas.
I also want to thank my parents Engelbert and Ursula Hucklenbroich and my wife
Silke Kirchen, who encouraged me to write this thesis and supported me in loving
care. They accompanied me throughout the very enjoyable and interesting time
during which I completed my thesis.
My special gratitude goes to Silke Kirchen for many scientific discussions, her
penetrating questioning of my results and her tireless commitment in the correction
of my manuscript.
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Wir leben nur so lange, wie wir neue Ideen haben und wie wir uns
bemühen diese neuen Ideen zu realisieren.
Erwin Ringel 1921-1994
Meiner Frau Silke und meinen Eltern
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Table of Contents
Glossary ..................................................................................................................................................... 6 1. Introduction ......................................................................................................................................... 7 1.1 Stem cells ........................................................................................................................................... 7 1.2 Neural stem cells .............................................................................................................................. 8 1.3 New approaches of stem cell based therapies ........................................................................ 12 1.4 Curcuma Longa ............................................................................................................................... 14 1.4.1 Curcumin ............................................................................................................................................... 14 1.4.2 aromatic-Turmerone ............................................................................................................................ 15 1.5 Scientific objectives ....................................................................................................................... 16 2. Methods and results ......................................................................................................................... 17 2.1 Publication ................................................................................................................................................ 17 3. Discussion ........................................................................................................................................... 27 3.1 General considerations ........................................................................................................................... 27 3.2 Risks of ar-Turmerone and curcuma longa therapies ............................................................ 30 3.3 Effects of other important molecules on NSCs and comparison to TUR ............................ 31 3.3.1 Endogenous molecules........................................................................................................................ 31 3.3.2 Control Experiment: Resveratrol ........................................................................................................ 36 3.4 Conclusion ........................................................................................................................................ 37 4. Summary ............................................................................................................................................. 38 5. Zusammenfassung ............................................................................................................................ 39 6. References .......................................................................................................................................... 41 7. Preliminary publications ................................................................................................................. 47 8. Lebenslauf .......................................................................................................................................... 48 5
Glossary
ar-Turmerone
aromatic Turmerone
BrdU
5-bromo-2'-deoxyuridine
CL
Curcuma longa
CNS
central nervous system
EGF
Epidermal growth factor
FGF2
Fibroblast growth factor 2
FGL
FG Loop
Fig.
Figure
i.c.v.
intracerebroventricular
iPS
induced pluripotent stem cell
MHC
major histocompatibility complex
mRNA
messenger RNA
MRT
magnetic resonance tomography
NCAM
neural cell adhesion molecule
NSCs
neural stem cells
OPN
Osteopontin (secreted form)
PBS
phosphate buffered saline
PCR
Polymerase chain reaction
PORN/FN
Polyornithine/Fibronectin
RES
Resveratrol
RG2
Rotorgene 2000 PCR analysator
RG3
Rotorgene 3000 PCR analysator
RNA
ribonucleic acid
ROS
reactive oxygen species
RT-qPCR
real time quantitative PCR
SVZ
sub ventricular zone
TUR
ar-Turmerone
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1. Introduction
1.1 Stem cells
The existence of stem cells was proposed first by the Russian histologist Alexander Maximov
in 1908. The first discovery of self-renewing cells was made by Till and McCulloch, who
found self-renewing cells in the bone marrow (71). Today the existence of stem cells is
undoubted. Within an adult body, stem cells are found at any places where tissue
replacement is necessary. Figure 1 illustrates potential uses of stem cells to treat diseases.
Stem cell proliferation is regulated by cytokines according to the requirements of the body.
After proliferation, the newly generated cells migrate to their target organ and differentiate
into the required cell type, where they replace aged or defective cells. These stem cells are
named adult stem cells or pluripotent stem cells. They are partially differentiated. Under
physiological conditions, these cells can only differentiate into cells downstream the same
pathway. For example, stem cells of the skin can only differentiate to skin-related cells, like
keratinocytes, but not into cells of other pathways, like brain cells or epithelial cells.
In this research line, neural stem cells from 14 days old rat embryos were used.
Fig. 1: The body uses adult stem cells to deal with a great number of diseases. These self-healing
approaches are not always effective, but new forms of therapy can support this body function.
Picture source: www.arizonapain.com
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Another group of stem cells are embryonic stem cells. They are the first, totipotent cells,
formed after fertilization of an egg cell. This cell type was first isolated from a mouse
blastocyst and put into culture by Evans and Kaufman in 1981 (15). Embryonic stem cells are
not found within an adult body. Working with these cells results in the destruction of the
embryo, which is inacceptable for ethical reasons regarding for example human derived stem
cells. Embryonic stem cells were not used within this research line.
1.2 Neural stem cells
The presence of neural stem cells (NSCs) in the adult brain was proposed in the late 1990s
(75). Later, methods were developed to isolate these cells from the brain, cultivate them in
vitro and to differentiate them into cells of lower hierarchy: neurons, astrocytes and
oligodendrocytes (65).
Fig. 2: Totipotent stem cells differentiate to pluripotent and neural stem cells (NSCs). NSCs are
found within the adult hippocampus and the sub ventricular zone, where they proliferate. They
migrate to their target and differentiate into new neurons, oligodendrocytes or astrocytes.
Picture adapted from www.arizonapain.com
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Today, it is known that NSCs (Fig.2) split into various subpopulations that are restricted to
certain areas of the brain, or developmental stages. These subpopulations are believed to
fulfil specific biological functions (41). During the last decade, magnetic resonance imaging
and positron emission tomography based methods were developed as non-invasive
techniques to study their function and fate in the living mammalian brain (7, 53, 66).
1.2.1 Normal stem cell function
NSCs proliferate in the sub ventricular zone (SVZ) and in the hippocampus (83) (Fig. 3).
Fig.3: In the adult mammalian brain NSCs are located within special compartments: the SVZ and the
hippocampus (red areas). After proliferation they migrate into the brain tissue to reach their target.
Picture copyright by Medi Visuals Inc.
Downloaded from (https://beyondthedish.wordpress.com/2014/02/07/).
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In the healthy brain, they are self-renewing and constantly produce new neurons (23). They
can protect the brain from central nervous autoimmunity and play a role in learning (22, 52).
NSCs can differentiate to astrocytes that respond to the inflammatory response in a variety
of CNS diseases and form an astroglial scar (21). They communicate with infiltrating
macrophages, which resolve the glial scar after a brain lesion, an interim stage in CNS repair.
Autoimmunity needs to be at equilibrium to maintain the integrity of the brain (Fig. 4). Fast
responding microglia as the brain’s resident immune cell act as antigen presenting cells and
activate invading T-cells (11). T-cells that present CNS antigens play a role in the recovery
from CNS injuries, a phenomenon that is named “protective autoimmunity” (32). NSCs
modulate the innate and adaptive immune system in the brain by closely interacting with
both microglia and T-cells (36, 74).
A second type of glial cells is derived from NSCs and is located closely to neurons. These
cells are called oligodendrocytes. They form myelin coatings (myelin sheath), which enhance
the transduction of neural signals. They protect, nutrify and support neurons in the brain
(28). Additionally, they play a role in establishing connections between neurons (76).
Fig. 4: CNS specific autoimmunity needs to be well balanced to maintain functional integrity of the
brain. Over-activation or loss of CNS-specific autoimmunity has a detrimental effect and is expected to
impair CNS integrity. Picture source: Schwartz (59) .
1.2.2 Stem cell function in CNS diseases
Neural stem cells play an important role in the response of the individual to diseases of the
brain. If lesions are present in the brain, the proliferation rate of endogenous NSCs is
increased and microglia become activated (Fig. 5) (83). This is also true for stroke, but NSCs’
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potential to self-renew is not enough to replace the tissue of the affected area (2). Invading
macrophages and activated microglia phagocytise the affected tissue (70). This is an
important function, because the glial scar, if not properly dissolved, is detrimental to healing
[14]. Elevated Osteopontin (OPN) levels, secreted by NSCs (26) and macrophages (63), can
be used as a prognostic marker for the outcome of stroke (40).
NSC dysfunction or loss due to radiation or chemotherapy in cancer treatment leads to a
long-term decline of cognitive functions like memory, attention, concentration and speed of
information processing [9]. Transplantation of NSCs into the brain leads to an improvement
of cognitive functions, as shown in a mouse model [10].
Fig. 5: Under normal conditions, microglia lie resident in the CNS and no macrophages can be found
in the parenchyma. Under pathological conditions, microglia become activated and macrophages
invade the parenchyma. NSCs enhance their proliferation rate and migrate to the injury where they
contribute to cell renewal and immune modulation. Antigen drainage to the lymph nodes and immune
cell mobilization is enhanced. Picture source: Schwartz (59)
In neurological disorders with ongoing inflammation due to e.g. autoimmunity, the beneficial
maintenance role of removing defective cells and cleaning of debris is misled and immune
cells attack normal tissue, which simply present a normal marker protein. Ongoing
neuroinflammation critically contributes to disorders like Alzheimer’s disease, multiple
sclerosis or Parkinson’s disease (5, 58, 59).
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For therapeutic use, NSCs can be propagated in vitro and genetically modified or loaded with
substances, which have anti-tumour effects. As NSCs tend to migrate to tumours inside the
brain, they can be used for targeted delivery of these drugs directly to a tumour (41).
1.3 New approaches of stem cell based therapies
Several diseases and aging lead to a loss of cells, which need replacement. New therapeutic
approaches, based on adult stem cells, can be divided into several groups:
1.3.1 Enhancing the survival rate of NSCs
NSCs are self-renewing in the adult brain, but their life time is limited. To enhance the
number of stem cells, therapies can be developed that aim at the prolongation of the stem
cell life (44, 57). A longer life span at a normal proliferation rate will end up with a higher
cell number within the proliferation zone. This approach has the advantage, that substances
that enhance the survival rate, but do not cause an increased proliferation have a lower risk
of contributing to the risk of developing cancer.
1.3.2 Enhancement of NSC proliferation
Enhancement of stem cells proliferation has a similar effect as the prolongation of their life:
After a defined period of time, more stem cells are present and can migrate into the brain
tissue and differentiate into neurons, oligodendrocytes or astrocytes to replace defective cells
(3, 30, 57). An increased proliferation rate bears the danger that NSCs do not have enough
time to repair gene defects and thereby accumulate mutations. An increased proliferation
rate may also lead to enhanced apoptosis, which counteracts the goal to enhance the stem
cell numbers, or to the generation of cancer cells, like stated above (73).
It is possible that drugs act in a two-fold way: by increasing proliferation and survival of
stem cells (57).
1.3.3 Guiding of stem cell migration
NSCs proliferate in the SVZ and the hippocampus (83). From their niche they migrate into
other parts of the brain, where they differentiate. This migration can be influenced be chemo
attractants like Osteopontin (77) or SDF-1 (68), or by physical influences like direct current
stimulation or strong magnetic fields (56, 61). This offers the physician a way to guide these
cells to the area of a lesion where they may contribute to tissue repair.
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1.3.4 Transplantation of stem cells
Stem cells, derived from the brain of fetal or adult mammals, can be extracted from the
brain and propagated in cell culture. These cells can be harvested and transplanted into or
close to a lesion in a recipient’s brain (54, 55). There they are able to differentiate into all
three cell fates, integrate into the brain structure and show long term survival (33, 50). It is
important to compare the MHC of the donor with the recipients MHC and to use only
compatible donors to support a long term survival of the transplant (79).
Regarding NSCs, there were hopes that transplanted stem cells could replace degrading
tissue in diseases like stroke, Parkinson’s or multiple sclerosis, but data implicit the
conclusion that the majority of transplanted cells only survive for some limited time within
the brain and rather have trophic effects like secreting cytokines, chemokines and growth
factors and protective effects from autoimmunity, but are not able to regenerate lesion areas
of the brain by replacing lost neurons (14, 37, 38, 52).
1.3.5 Induced pluripotent stem cells (iPS cells)
In the last years, a new type of stem cell has come into focus. The so called iPS cells are
pluripotent cells, which have been generated by reprogramming of cells downstream the
differentiation line. This reprogramming is accomplished by retroviral transduction of the
‘Yamanaka factors’ Oct4, Sox2, Klf4, and c-Myc. For generation of iPS cells, cells derived
from adipose tissue have been reported as being good candidates. These cells are capable to
differentiate again to all three germ lineages, mesoderm, ectoderm and endoderm after their
formation (69). Cells of the mesoderm differentiate to muscle, red blood, and kidney tubule
cells, endoderm cells form alveolar, thyroid, and pancreatic cells, and ectodermal cells
become skin, neurons, and pigment cells.
Retroviral transduction must always be done with great care, as viral genes are cotransfected, possibly leading to an unwanted outcome. For this reason especially designed,
very safe vectors, capable of self-deleting viral genes after transduction were designed
recently (16).
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1.4 Curcuma Longa
Curcuma longa (CL) (Fig. 6) is a plant from the family of Zingiberaceae. The Curcuma family
hat about 90 different species. The most familiar plant of this family is Ginger (Zingiber
officinale). The natural habitat of the plant is from Asia to northern Australia, but it is
cultivated in many tropical regions. The plant grows up to one meter in height and has
highly branched, orange coloured rhizomes. CL needs temperatures between 20°C and 30°C
to grow. Because of the orange colour of the root, it was first used as a dye. Plants of the
curcuma family have been used in traditional Chinese and ayuverdic medicine for thousands
of years (19). Recently, CL tuber powder was used to create silver nanoparticles from
aqueous silver nitrate in an eco-friendly way because of its reductive and stabilizing
properties (60).
Fig. 6: Extracts from the Curcuma longa plant accounts for the yellow color in curry powder. It has
been used for centuries in traditional Indian and Chinese medicine. It is known for its neuroprotective
capacities. Over the last decades, CL extracts are being studied for their properties to treat
neurological diseases like Alzheimer’s, Parkinson’s or dementia.
In Europe curcumin, an extract of the CL root, is known as an ingredient of curry powder
and it is the ingredient accounting to the yellow colour of the spice.
1.4.1 Curcumin
Curcumin is the most important and most studied bioactive compound, derived from the
curcuma plant. Its properties as a drug candidate have been studied excessively. Its
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molecular structure is displayed in Fig. 7. Curcumin has anti-inflammatory, anti-proliferative
and anti-apoptotic effects [23-26]. Curcuma longa is also known to possess neuroprotective
effects and can prevent death of mice after bites of the South American rattle snake
(Crotalus durissus terrificus) [27].
Fig. 7: Molecular structure of Curcumin, the most studied component of the curcuma plant.
Picture source: Sigma Aldrich website
Administration of curcumin has anti-tumour effects, which are enhanced in visible or UV-A
light. It increases apoptosis rates in tumour cells and ameliorates Cisplatin induced
ototoxicity. Additionally, curcumin has a positive effect in experimental rat autoimmune
neuritis (6, 10, 18, 24). Unfortunately, curcumin has a low solubility in water, so formulations
must be developed to enhance the bioavailability of curcumin.
1.4.2 aromatic-Turmerone
The major component of the essential oils of the Curcuma longa plant, which is known as
Turmeric in English speaking countries, is ar-Turmerone (Fig. 8). Other components are
turmerone and curlone. These three components make up to 50-60% of the solvent extract
from the CL root. They are stable at different temperatures, but are sensitive to light (27).
TUR can be desorbed after oral (82) or i.p. administration and passes the blood brain barrier
(49).
Fig. 8: Molecular structure of ar-Turmerone ( (S)-2-Methyl-6-(4-methylphenyl)-2-hepten-4-one)
Source: Sigma Aldrich website
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According to Ferreira et al., the antivenom effects of Curcuma longa does not belong to
curcumin, but to the essential oils of the plant, mainly consisting of ar-Turmerone [23, 24].
The authors additionally report an anti-proliferative effect of this oil on human lymphocytes
[23].
In 2008, Dohare et al. reported a neuroprotective effect of the Curcuma oil in a stroke model
of rat. The group stated a smaller lesion size of an experimental stroke, measured by MRI,
and an amelioration of ischemia-induced neurological deficits and brain oedema volumes. By
western blot analysis, they showed the correction of inflammatory, apoptotic and oxidative
stress markers due to administration of Curcuma oil and an increased survival rate of
neurons (13).
In 2010, Yue et al. reported an anti-proliferative effect of CL crude extract on human cancer
cells and a stimulatory effect on the proliferation of human peripheral blood mononuclear
cells (81).
Still, formerly unknown effects of TUR are being discovered. Recently, anti-convulsive effects
of TUR have been found, making it a drug candidate for epilepsy [34].
Stroke causes a characteristic neuroinflammatory response involving the activation of
microglia (also compare chapter 1.2). Due to these inflammatory processes, cells are
affected that were not located within the original ischemic area, widening the damage to the
patient’s brain [33]. Park et.al. reported in 2012 the anti-inflammatory effect of arTurmerone on amyloid beta stimulated microglia, and suggested the possible use of the
substance as therapeutic agent for the treatment of neurodegenerative disorders, also
associated with neuroinflammation (47).
1.5 Scientific objectives
Aim of this study was the identification of chemicals that either enhance the life-span of
NSCs or increase their proliferation rate. For this purpose, Osteopontin (OPN), Resveratrol
(RES) and ar-Turmerone (TUR) were chosen for investigation. As the effects of RES on NSCs
were already studied (45, 67, 84, 85) this substance was chosen as a control for the validity
of the experimental setup. Some promising data were available about the effects of OPN on
NSCs (30, 39, 72, 77) that needed further investigation. Nothing was known about TUR
mediated effects on NSCs. OPN and TUR were evaluated in vitro and in vivo, while RES was
only evaluated in vitro.
As described above, ar-Turmerone exhibits neuroprotection in a rat stroke model and antiinflammatory effects on microglia. These findings make TUR a possible candidate for
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treatment of stroke that needs further studies. Up to now, nothing was known about the
direct effects of TUR on neural stem cells. These effects were evaluated in the present study
and compared to the effects of the other investigated neuroprotective substances
Osteopontin and Resveratrol.
2. Methods and results
2.1 Publication
Aromatic-turmerone induces neural stem cell proliferation in vitro and in
vivo
Joerg Hucklenbroich, Rebecca Klein, Bernd Neumaier, Rudolf Graf,
Gereon Rudolf Fink, Michael Schroeter and Maria Adele Rueger
Stem Cell Research & Therapy 2014, 5:100
http://stemcellres.com/content/5/4/100
doi:10.1186/scrt500
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3. Discussion
3.1 General considerations
Choice of stem cell populations for in vitro culture
To test proliferation and differentiation properties of stem cells that can be found within the
brain, neural stem cells are necessary. Stem cell systems like iPS or embryonic stem cells
were unsuitable cell models due to their differentiation properties. They are upstream the
differentiation cascade of NSCs or artificial stem cells and are able to differentiate into cell
types other than the three types NSCs differentiate into: astrocytes, neurons and
oligodendrocytes.
NSCs can be isolated from the SVZ of adult rats and of embryos. To obtain a sufficient
number of NSCs, 14 days old rat embryos were chosen as donor organisms. As starting
culture, cells from eight to twelve individuals were pooled. Cells were harvested from the
SVZ by surgery and sheered by pipetting to obtain single cells. These cells were seeded to
Petri dishes, coated with poly-ornithine and fibronectin. Due to media conditions, only NSCs
survive the first passage, other cells like neurons, oligodendrocytes or glial cells and
fibroblasts from the tissue adhere to the plate too strongly for passaging, or die due to
insufficient media conditions. We regarded the cell population from the second passage as
suitable for in vitro experiments. As determined by SOX2 staining, these cell populations
contain about 90% of undifferentiated NSCs (26). Although it would be possible to obtain
higher percentages of NSCs by using commercial kits like the Anti-PSA-NCAM MicroBeads kit
(Miltenyi Biotec, Bergisch Gladbach, Germany) we regarded the risks of such techniques
(influencing the cells by adding antibodies and magnetic separation) higher than the risks of
a few already differentiated cells present in the culture. Especially, we wanted to avoid
strong magnetic fields, as it is known, that these fields influence neural cells (9, 64).
Cells for experiments were used up to an age of a maximum of five passages, as older cells
tend to start differentiation even in the presence of FGF. Cells, frozen from passage two or
three, were used for no longer than two passages. Cultures showing signs of differentiation
were discarded, as cells showing these signs are already committed to a differentiation route
and therefore can no longer be influenced by treatment with substances. Our method of
harvesting the cells from the Petri dishes was very gentle and avoided enzymatic stress of
the cells by trypsin: we removed the culture media and added PBS for 20 min at 37°C. After
incubation, the cells disconnected from the dish and were harvested by using a 10ml pipette,
concentrated by centrifugation, resuspended, counted and seeded to new culture dishes.
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Cells were propagated at 37°C in presence of 5% (v/v) CO2. Other methods, like
trypsinisation or using a cell scraper, lead to stress, due to cellular damage, which probably
would lead to higher intracellular OPN levels. As OPN induces survival, high levels could
possibly hinder the detection of other test substances in the search for survival, as the
pathway could already be saturated.
Culture of NSCs
Culturing cells is always an artificial system. Under culture conditions cells grow within a
community, which does not exist in nature. Cultures of NSCs consist of NSCs and a few
additional cell types, which were generated during the culture by differentiation. Under
physiological conditions within the brain, these cells grow in close contact to differentiated
neural cells and other cells like e.g. cells from the vascular system or blood cells. They
interact with these cells; secrete signal proteins, which most likely differ from those within a
culture dish. There is always a necessity to confirm results in a natural environment: a living
organism.
NSCs can be cultured either floating in the media and growing into neurospheres, or
adhering to the culture disk. If culture plates are coated with poly-ornithine and fibronectin
(PORN/FN), cells adhere to the plates and are able to migrate there, if plates are uncoated,
cells adhere to each other and grow into neurospheres. An advantage of neurospheres is the
close distance of higher number of cells to each other, which makes them benefit from
cytokines produced by neighbouring cells. A disadvantage of this system is that cells,
residing in the centre of the neurosphere, are not optimally supplied by oxygen and
nutrients, due to the longer diffusion route. This alters their differentiation route in favour to
glial differentiation and increases their migrational activity (43). Due to these major
disadvantages we chose to grow the cells as a monolayer by coating the culture dish with
PORN/FN.
Precaution is necessary on designing studies, where TUR is used to enhance the proliferation
rate of stem cells in culture to generate cells for transplantation. It must be considered that
transplanted cells have proliferated in vitro, and were exposed to chemicals and cytokines
like FGF2 or EGF, and passed more cell cycles than they would have under physiological
conditions. During culture they could have already differentiated partially which could alter
the cells’ response when re-introduced in vivo. An example for this risk is the finding that
neurogenic precursors that were cultured in the presence of EGF confer an invasive, stem
cell like behaviour (12). Additionally it must be taken into consideration that these cells are
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taken from another organism; they bear the risk of forming abnormal circuitry that may
contribute to brain malfunction like abnormal behaviour or epilepsy.
Route of application for bioactive molecules
From a patients view, the least invasive way to administer a drug is the best way. A patient
would prefer oral or transdermal application. Injections bear the risk of infections and
injections directly into the brain will damage tissue and always lead to inflammatory
processes.
A researcher wants the test substance instantly at the target organ in a concentration high
enough to see an effect. Orally or transdermally applied molecules have to pass organs,
where they could be modified, like the liver, before they reach the target. They even may be
cleared from the organism before reaching the target in significant concentrations.
Additionally it must be considered, that the test molecule may not pass the blood brain
barrier.
In our first in vivo experiment with TUR, rats were treated by i.c.v. application to ensure
that it reached the target cells within the SVZ in sufficient concentration and without possible
modifications by passing the recipients metabolism. Despite these advantages, this nonphysiological application route has some disadvantages: it is tedious, causes very high drug
concentrations in small areas, and parts of the brain are damaged by the injection needle
(20). Later, we learned that TUR can be administered orally and passes the blood brain
barrier (49). Additional experiments with oral administration of TUR are recommended.
Especially dose escalating studies are recommended to find the best dose to enhance
proliferation while minimizing toxic effects.
Behavioural tests
Diseases like stroke cause behavioural anomalies. Motor deficits of laboratory animals can be
studied in behavioural tests like the catwalk. The footprint of a rat suffering from stroke,
running in the catwalk, differs from a healthy animal. They set their feet with less strength
than healthy animals. This can be measured and gives data on the severity of the
neurological disorder. In vivo we confirmed the in vitro results of NSC proliferation by PET
imaging as well as by histology. Therefore, we expect that the enhanced proliferation of
NSCs after TUR medication also would lead to a faster recovery of rats with experimental
stroke, as brain repair would accelerate by the higher number of NSC. This hypothesis will
need to be tested in a behavioural paradigm on stroke rats, e.g. using the catwalk system.
Another idea is to study the performance of an Alzheimer’s rat or mouse model in the water
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maze test under the influence of a TUR based therapy. It would be interesting to see, if a
combination of TUR and OPN have synergistic effects, as OPN administration caused longer
survival of NSCs (see results below).
3.2 Risks of ar-Turmerone and curcuma longa therapies
Risks of TUR based therapies
An increase of NSCs proliferation rate has the effect that more cells are present to repair
lesions in the brain, but it must be taken into consideration that excessive proliferation of
stem cells bears the danger of mutations to accumulate, possibly leading to brain tumours,
e.g. neuroblastoma (80). This danger is even higher as TUR inhibits proliferation of
lymphocytes and the activity of natural killer cells, which have a cytotoxic effect on tumour
cells (17).
Despite these concerns, no reports of mutagenic effects of TUR or other substances present
in the curcuma longa plant have been published yet. In contrast, many reports were
published proposing an anti-tumour effect of CL or TUR (4, 29, 31, 35, 42, 46, 48). The long
tradition of Indian and Asian people to use the curcuma plant as spice and in traditional
medicine (17) confirms the safety of its use. Additionally toxicological and mutagenic studies
of the Curcuma oil have shown neither toxic nor chronic effects (34).
Therefore it is probable that a TUR based therapy is not likely to increase the patients risk to
develop brain tumours.
Adverse effects of CL and CL products
Many beneficial effects of Curcuma and extracts of the Curcuma plant have been reported,
but a literature screening for adverse or even harmful effects brought almost no result. The
only report found was from Calapai et.al., who reported a contact dermatitis after topical
application of CL (8). This finding was surprising, as the anti-inflammatory effects of CL are
well known and Agarwal et.al. recently reported the topical use of CL to suppress LPS
induced inflammation in rats (1). Possibly, it was an effect of a high dose of the substance.
30
3.3 Effects of other important molecules on NSCs and
comparison to TUR
3.3.1 Endogenous molecules
Osteopontin (OPN)
TUR is a secondary metabolite of the CL plant and cannot be produced within a patient’s
brain. In contrast, OPN is naturally produced within the body and it had to be evaluated
whether the target cells themselves produce this molecule.
We found by gene expression analysis on mRNA level that NSCs themselves express OPN.
OPN was strongly expressed after passaging of the cells and the level decreased with culture
time (Fig. 9). Additionally, OPN was up-regulated, if the cells were exposed to nonphysiological conditions, like elevated CO2 levels (26).
Fig. 9: OPN mRNA content of NSCs in relation to the culture time. After plating, the OPN mRNA was
at the highest level, decreasing with time. Data were generated in two experiments and evaluated
with two different PCR machines. The OPN gene expression level was normalized against the
reference genes HPRT and RPL13a.
These findings led to the conclusion that OPN acts as a stress related protein within NSCs.
This conclusion is supported by Mendioroz et.al. who reported an increase in the OPN level in
correlation to the severity of stroke (40). Possibly higher doses, close to the toxic doses, of
TUR will also increase the OPN production of NSCs and must be taken into consideration.
OPN is also believed to induce the migration of NSCs (39, 77, 78). As OPN is up-regulated by
passaging of the NSCs, we studied the migration of NSCs under the influence of OPN
treatment at different doses at day 1 and day 3 after seeding the cells into the migration
chamber (Fig. 10).
31
Fig. 10: After overnight migration time, migrated cells were stained with crystal violet and extracted
with acetic acid. OD560 was measured to determine the relative amount of cells migrated. Within this
migration assay, no migration enhancement was visible after treatment with OPN at day 1 after
plating (left side). At day 3 after plating (right side), OPN treatment enhanced NSCs migration
between a concentration of 1.56 µg/ml and significantly at 3.125 µg/ml (p=0.076).
No migration was visible at day 1 after plating; in contrast, the migratory activity seemed to
be reduced. At day 3, a migratory effect could be seen at OPN doses between 1.6 and 3.1
µg/ml. At 3.125 µg/µl this effect was significant (p=0.039), the lower 1,56 µg/µl dose needs
more data (p=0.076). This led to the conclusion that a culture time of about three days was
necessary to reduce the effects of endogenous OPN on the results of NSC experiments. The
enhancement of migration by OPN was confirmed within an RT-qPCR experiment targeting
the migration marker CXCR4 and by histological analysis in a NSC culture by using a CXCR4
antibody and comparison to controls (data not shown). As OPN only promoted migration in a
small therapeutic window, the internal OPN concentration of a culture must be considered if
does escalating studies are performed. Especially, it must also be considered that the OPN
concentration in patients suffering from stroke are already higher, to avoid an adverse effect
on NSCs migration.
Up to date, we have not investigated the effect of TUR on the migration of NSCs. It can be
speculated that TUR will not increase migration, as stem cells migrate to a target, where
they will differentiate. Proliferation as seen after TUR treatment probably rather takes place
in cells that do not migrate.
To be able to analyse the effects of TUR on the proliferation of NSCs, we additionally
determined if OPN had an increasing effect on the proliferation rate of NSCs, as stated by
Kalluri et.al. (30). In contrast to Kalluri et.al. we found that OPN did not enhance the
proliferation rate of NSCs, but the survival rate of NSCs in the presence of FGF2 (Fig. 11). A
reason for this difference may be that Kalluri et al. used NSCs derived from adult
32
hypertensive rats, growing in neurospheres, while we cultured NSCs growing adherent from
E14 embryos. A neurosphere is a non-adherent spherical cluster of cells, where cells,
growing at the centre of the sphere are exposed to lowered oxygen and enhanced CO2 levels
due to a longer diffusion time of gases within the cell clump, resulting in an already
enhanced OPN expression of these cells, due to cellular stress (26).
Fig. 11: NSCs were cultured for 24h in an adherent cell culture system and exposed to increasing
concentrations of OPN. Their proliferation rate was determined after 24h culture time and BrdU
administration by immunofluorescent staining and cell counting. No significant increase of the
proliferation rate could be determined.
Our finding that OPN did not increase proliferation suggested that the proliferative effect of
TUR was not caused by toxic effects on the NSCs, resulting in higher OPN levels.
To understand a TUR-based effect of the differentiation of NSCs, we needed to know if the
presence of elevated OPN levels within the culture altered the differentiation of the NSCs.
This was evaluated at a concentration of 6.25 µg/ml OPN for a period of 10 days (Fig. 12).
33
Fig. 12: NSCs were exposed to 6.25µg/ml OPN and adherently cultivated for 10 days with FGF2
withdrawal. Differentiation was evaluated by immunofluorescence staining and counting. No
significant effect of OPN was seen compared to control.
No significant effect of OPN could be determined on the outcome of the differentiation of the
NSCs. Thus, the increasing number of new neurons under the influence of TUR could not
have been caused by stress-mediated enhanced OPN levels.
The last parameter to be evaluated was, if OPN increased the survival rate of the NSCs in
culture. This was evaluated in vitro by culturing the cells under influence of increasing doses
of OPN (Fig. 13).
Fig. 13: NSCs were cultivated under influence of increasing doses of OPN. Cells were photographed
after 5h, 22h and 30h incubation time and counted manually or by using the Image J (ImgJ) program
with a threshold of 20 pixels. No difference between manual and automated counting could be seen.
A significantly increased survival rate (p= 0.00011) of NSCs was seen after 30h of incubation time at
1.25 µg/ml compared to control.
34
During the first 22h of incubation, no effect of OPN was visible, but at 30h doses between
1.25 µg/ml and 12.5 µg/ml significantly increased the survival rate of the cells. Higher doses
than 12.5 µg/ml ameliorated the survival effect and no difference to control was visible.
These data were supported by in vivo experiments on naïve rats and rats suffering of an
experimental stroke provoked by administration of Rose Bengal and photo thrombosis
(Fig.14).
Fig. 14: Determination of new NSCs after OPN administration into the ventricle within the SVZ of rats
suffering from experimental stroke, by BrdU administration and histological staining. The number of
BrdU positive cells was higher in animals that received OPN compared to animals that received saline
as sham injection.
BrdU is a nucleotide base analogue that is incorporated in dividing cells and replaces
thymidine in the DNA, thereby marking proliferating cells (51). All animals which received
OPN treatment showed a higher number of BrdU positive cells within their SVZ, regardless of
the presence of an experimental stroke. Additionally the size of the SVZ of OPN treated
animals was wider than that of control animals (26). As the cell number of a tissue increases
regardless a possible proliferative effect or survival effect after a period of time, these
experiments confirmed the results from the in vitro studies.
Conclusion of OPN results for TUR experiments
These results allowed us to screen for new molecules and their abilities to enhance the
proliferation rate or to alter the differentiation outcome regardless of their OPN level due to
culture conditions or their possibly increased stress level due to oxygen depletion during cell
isolation or toxic effects of the test substance. The longer survival rate of OPN-treated cells
35
needs to be taken into consideration when investigation possible drug candidates, but no
enhanced survival rate was found after TUR application.
3.3.2 Control Experiment: Resveratrol
Resveratrol (RES)
Resveratrol, naturally occurring in red wine, has been studied for a long period and is
classified to act as a neuroprotective agent, by acting anti-apoptotic by counteracting ROS
formation, activation of the SIRT-1 pathway (62), and acting anti-inflammatory (84, 85).
Additionally, Hauss et.al. report an induction of differentiation of NSCs into mature neurons
(25). The anti-apoptotic effects have been challenged by Park et.al., who reported the
inhibition of NSC proliferation, and a lower performance of RES treated mice in the water
maze test. Additionally he challenged the SIRT-1 induction by RES (45).
If RES had an anti-apoptotic effect on NSCs, a longer survival rate should be detectable, but
no enhanced proliferation should be visible within the culture.
To clarify the differences in the reports of RES activity on NSCs and to proof the validity of
our test system, we used RES in a ten days differentiation study of rat NSCs in vitro with
immunofluorescent staining and cell counting (Fig. 15).
Fig. 15: Left side: Percent of cellular fates after 10 days of differentiation under influence of
6.25 µg/ml Resveratrol. No clear tendency of a specific cell fate was visible. Right side: The total
number of cells counted within this experiment was much higher under the influence of RES compared
to the control group.
Our results did not give a clear tendency for a preferred differentiation into a specific fate,
but the total number of cells, surviving the 10 days of differentiation after FGF2 withdrawal,
was significantly higher within the RES treated cultures (n=2; p=2.4*10-7 and P=1.4*10-5).
36
Because all cells, counted within this experiment, did belong to either one of the two groups,
and the trial was repeated only once the calculation of a standard deviation is not feasible.
Therefore, we suggest that RES might have an anti-apoptotic effect on differentiated or
partly differentiated cells, and that our test system showed this effect.
To our surprise, the fraction of undifferentiated cells were not found to be lower in the REStreated group, as determined by SOX2 antibody staining, although cells looked more
differentiated, even after 30h treatment with RES in a survival experiment (Fig. 16).
Fig. 16: NSCs treated with 6.25 µM RES looked more differentiated after 30h of incubation under a
phase contrast microscope, compared to a control group.
These results thus did not give a clear picture of the action of RES on the differentiation of
neural stem cells. It seemed possible that the RES-treated cells rest within a partially
differentiated state, but are still able to proliferate at a decreased rate (data not shown),
even after 10 days of FGF2 withdrawal. More research is needed to clarify this thesis.
3.4 Conclusion
Our results show for the first time that TUR significantly enhances proliferation of NSCs.
Additionally, the substance altered the differentiation of the stem cells in respect to an
increase of neurons at a dose of 6.25 µg/ml, while the differentiation properties of NSCs
treated with OPN remained unaltered. The percentage and total number of TUR-treated
stem cells was significantly reduced after 10 days of differentiation induced by FGF2
37
removal, compared to the control group, and there was a trend to a lower percentage of
astrocytes, but this finding was not significant at the dose tested.
4. Summary
Ar-Turmerone and Resveratrol are secondary metabolites from herbal origin. Osteopontin is
produced naturally in the body.
In this study, the effects of ar-Turmerone on neural stem cells in vitro and after
intraventricular injection into the rat brain in vivo were examined. The in vitro effects were
compared to the effects of Osteopontin and RES.
It was found that TUR enhanced the proliferation rate of neural stem cells in vitro and in
vivo, while OPN did not enhance the proliferation rate, but the survival rate of NSCs in
culture and in vivo, and enhanced their migration in vitro. Under the influence of TUR, NSCs’
differentiation properties were altered, in respect that they tended to differentiate into
neurons, while OPN did not alter their differentiation properties. The number of NSCs under
influence of TUR after 10 days of differentiation was significantly reduced in comparison to
control. NSCs treated with RES preferably differentiated to become astrocytes. In contrast to
these TUR and OPN results, RES caused an early differentiation of NSCs, but the survival
time of the differentiated cells in culture was much higher than the survival time of a control
culture or of a culture treated either with TUR or OPN.
These findings make all three biomolecules interesting candidates for a variety of
neurological disorders.
38
5. Zusammenfassung
Im adulten Gehirn von Wirbeltieren sind neuronale Stammzellen vorhanden. Diese spielen
eine Rolle beim Lernen, beim Ersatz defekter Zellen und bei Reparaturvorgängen, zum
Beispiel nach einem Apoplex.
Es besteht die Hoffnung durch eine Verlängerung der Überlebensdauer dieser Zellen, eine
Verstärkung ihrer Proliferation oder durch eine zielgerichtete Migration den Heilungserfolg
bei neuronalen Defekten zu verbessern.
Curcuma longa oder Gelbwurz ist eine Pflanze aus der Familie der Ingwergewächse. Der
Hauptbestandteil des aus ihr gewonnenen Öles ist aromatisches Turmeron (TUR). Dieses Öl
ist bekannt für seine neuroprotektive Wirkung bei einem experimentell herbeigeführten
Schlaganfall der Ratte. Zudem überleben mit Kurkuma behandelte Mäuse eine Injektion von
Toxin der südamerikanischen Klapperschlange, deren Gift hauptsächlich aus Neurotoxinen
besteht. Weitere bekannte Wirkungen dieser Substanz sind eine Hemmung der Proliferation
und Förderung der Apoptose von Krebszellen, eine Förderung der Proliferation peripherer
mononuklearer Zellen, krampflösende Effekte, sowie entzündungshemmende Eigenschaften.
Ziel dieser Forschungsarbeit war die Untersuchung der Wirkung von TUR auf neuronale
Stammzellen.
Hierfür wurden neuronale Stammzellen aus 14 Tage alten Rattenembryonen gewonnen, in
vitro kultiviert und vermehrt. Zellen, nicht jünger als Passage zwei und nicht älter als
Passage vier, wurden für Experimente eingesetzt.
Im Proliferationsexperiment, bei dem sich teilende Zellen mit Hilfe des Nukleotidanalogons
BrdU markiert werden, konnte gezeigt werden, dass unter dem Einfluss von TUR eine
signifikante Steigerung der Replikationsrate im Vergleich zur Kontrolle bewirkt wurde. Dieser
Befund wurde in einem dafür neu etablierten, quantitativen Echtzeit PCR-System durch
Nachweis der Steigerung des intrazellulären Gehaltes von mRNA des als Proliferationsmarker
bekannten Gens Ki67 bestätigt.
In einem Differenzierungsexperiment mit 10-tägiger Dauer wurden neuronale Stammzellen
durch Entzug des Mitogens FGF2 zur Differenzierung angeregt. Histologisch konnte mit Hilfe
einer Antikörperfärbung auf das Zielprotein TuJ1 gezeigt werden, dass die neuronalen
Stammzellen unter dem Einfluss von TUR bevorzugt zu Neuronen differenzierten. Die
Gesamtzahl der nach 10 Tagen überlebenden, undifferenzierten Zellen war jedoch im
Vergleich zur Kontrolle signifikant erniedrigt.
39
Die proliferationsfördernde Wirkung einer einmaligen, intraventrikulären TUR Injektion
konnte zudem in vivo im Tierexperiment an gesunden Ratten, mit Hilfe der PositronenEmissions-Tomographie, sowie histologisch, bestätigt werden.
Die mit TUR gewonnen Befunde wurden in weiteren Experimenten mit der Wirkung der
körpereigenen Substanz Osteopontin (OPN) sowie des aus Weintrauben gewonnen
Resveratrols (RES) verglichen, da OPN und TUR Effekte auf neuronale Stammzellen aus der
Literatur bekannt waren, jedoch weitere Versuche zur Abklärung der Wirkung nötig waren.
Im Gegensatz zu TUR konnte bei den anderen Substanzen keine proliferationsfördernde
Wirkung nachgewiesen werden. Jedoch zeigte sich bei OPN eine signifikant verlängerte
Überlebensrate der neuronalen Stammzellen.
Außerdem konnte gezeigt werden, dass neuronale Stammzellen Osteopontin produzieren.
Eine Erhöhung des intrazellulären m-RNA Gehaltes für Osteopontin durch Stressfaktoren wie
Passagierung der Zellen oder erhöhtem CO2 Gehalt der Umgebungsluft während der Kultur
wurde mittels PCR gezeigt. Eine vermutete Steigerung des Migrationsverhaltens von
neuronalen Stammzellen durch Behandlung mit Osteopontin konnte zunächst nicht bestätigt
werden. Eine dreitägige Vorinkubation der Zellen in der Untersuchungskammer und eine
damit verbundene Herabregulation des intrazellulären OPN Gehaltes ermöglichte in weiteren
Experimenten jedoch den Nachweis eines verstärkten Migrationsverhaltens im Vergleich zur
Kontrolle. Dieser Befund konnte mit Hilfe der PCR auf den Migrationsmarker CXCR4 auf mRNA Ebene, sowie durch Immunfärbung auf das Membranprotein CXCR4, bestätigt werden.
Die Testsubstanz RES bewirkte in vitro eine Veränderung des Differenzierungsverhaltens der
Zellen in Richtung Astrozyten, was aus der Literatur bekannt ist und als Kontrollexperiment
diente. Zudem konnte gezeigt werden, dass RES-behandelte Zellen auch bei niedriger
Dosierung zu verstärkter Differenzierung neigten. Diese differenzierten Zellen zeigten dann
jedoch unter dem Einfluss von RES eine deutliche Verlängerung der Überlebensrate der
differenzierten Zellen im Vergleich zur Kontrolle und zu mit den anderen beiden Substanzen
behandelten Zellen.
Die gezeigten Befunde machen alle drei Substanzen zu interessanten Kandidaten für
Medikamente zur Behandlung einer Vielzahl neurologischer Erkrankungen.
40
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7. Preliminary publications
Aromatic-turmerone induces neural stem cell proliferation in
vitro and in vivo
Joerg Hucklenbroich, Rebecca Klein, Bernd Neumaier, Rudolf Graf, Gereon Rudolf
Fink, Michael Schroeter and Maria Adele Rueger
Stem Cell Research & Therapy 2014, 5:100
http://stemcellres.com/content/5/4/100
doi:10.1186/scrt500
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8. Lebenslauf
Mein Lebenslauf wird aus Gründen des Datenschutzes in der elektronischen
Fassung meiner Arbeit nicht veröffentlicht.
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