Integration of embryonic neuroectodermal stem cells
into brain tissue: studies on implanted neural stem cells
PhD thesis
Kornél Demeter
Institute of Experimental Medicine
of the Hungarian Academy of Sciences
Laboratory of Neural Cell- and Developmental Biology
Semmelweis University
Molecular Medicine Doctoral School
Cellular and Molecular Physiology Program
Supervisor: Dr. Emília Madarász, C.Sc.
Official opponents:
Dr. József Takács, C.Sc., Ph.D.
Dr. Lajos László, C.Sc., Ph.D.
Chairman of examination committee: Dr. Anna Faragó, D.Sc.
Members of examination committee: Dr. András Váradi, D.Sc.
Dr. Emil Monos, D.Sc.
Budapest, 2006
Introduction
Implantation of neural stem cells into the brain is regarded as a potential
therapeutic tool for cell replacement and gene delivery for damaged regions of the
central nervous system. Local production of some missing factors, such as
neurotransmitters, enzymes or growth factors achieved by grafting specially engineered
cells was demonstrated to improve or reconstitute some of the targeted functions. In
order to achieve long-lasting clinical improvements, the grafted cells not only have to
survive in the recipient tissue, but they also have to integrate anatomically and
functionally. Both survival and integration require the existence of coordinated
interactions between the grafted cells and the host tissue.
An intriguing question is that why the adult brain is incapable for recovery, if
neural stem cells (with the capacity of neuron and glia production) are present for the
whole life-span. We put two main assumptions:
1. Stem cells persisting in the adult brain represent already committed populations,
and thus, they can produce only some limited types of cells, not enough for tissue
recovery.
2. As an alternative, stem cells of the adult brain have wide developmental potential,
but the host environment inhibits some routes of differentiation because:
i. only limited cell types can survive in the adult brain environment and/or
ii. the molecular compounds in the adult brain tissue direct the differentiation to
some restricted developmental routes.
In the last few years, intensive efforts have been made to find the right types of
cells for grafting. Neural progenitors derived from embryonic brain tissue, embryonic
stem or teratocarcinoma cells, bone marrow-derived stem cells, adult neural stem cells
and immortalized neural progenitors were shown to integrate in various regions of the
CNS. Neural stem cell populations derived from different brain regions and from
different developmental stages exhibit different phenotypic characteristics and
developmental potential. Correspondingly, they may need different conditions for
survival, proliferation and differentiation.
In our recent studies, the effects of the tissue environment on the fate of
neuroectodermal stem cells were analysed by implanting pheno- and genotypically
identical populations of stem cells (NE-4C) into normal and pathophysiologically
altered mouse cortices.
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NE-4C cells were cloned from primary brain cell cultures prepared from the foreand midbrain vesicles of 9-day old transgenic mouse embryos lacking functional p53
"tumour suppressor" protein. NE-4C cells divide continuously if maintained under
normal tissue culture conditions and display several characteristics of non-differentiated
neural stem cells. In the presence of all-trans-retinoic acid (RA), however, these cells
differentiate into neurons and astrocytes through well-defined stages with specific
morphological, molecular and cell physiological characteristics.
In order to investigate the intracerebral fate of implanted stem cells, sub-clones of
NE-4C cells expressing either the green fluorescent protein (GFP) or the heat-resistant
placental alkaline phosphatase (PLAP) were established. Cells with stable production of
the marker proteins and maintaining the characteristics of the mother line were
implanted into the forebrains of adult, newborn and foetal mice, as well as into lesioned
or tumour-invaded brain areas of adult pathophysiological model mice. Occasionally,
cells were pre-labelled also with bromo-deoxyuridine (BrdU) for evaluating the rate of
their proliferation.
Inside the CNS, the fate of the implanted cells depends not only on the
developmental potential and characteristics of the grafted cells, but largely, also, on the
cellular and extracellular surroundings provided by the host locus. To investigate the
influence of the host environment, the cells were implanted into the forebrains of
healthy animals at different ages and into pathological model-animals.
According to data obtained in several laboratories, post-apoptotic conditions may
turn the adult somatosensory cortex receptive for embryonic neurons and neuronal
precursors. The appearance of several embryonic tissue characteristics under these
conditions together with our previous results on the ready integration of NE-4C cells
into the early embryonic chick brain encouraged us to implant these cells into apoptotic
brain areas. To induce extended apoptotic zones in adult mouse cortices, a cryogenic
lesion model was used. By this method, necrotic core regions and peri-lesion rims with
reproducible size were obtained in the parietal cortex.
As a novel tool to reach and destroy brain tumours, the use of engineered neural
stem cells (NSCs) as vehicles for delivery of anti-tumour agents was suggested.
According to some promising results, NSC-like cells may migrate to and invade
neoplastic tissues if implanted into the brain or delivered through intravascular
injection. The effects of potential tumour-derived factors and the environment in and
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around brain tumours are largely unknown. The wide variety of tumour-types and the
different invasiveness of different glioma cells, however, indicated that distinct
glioblastomas may provide different conditions for stem cell migration and survival.
The available data suggested that tumour-targeting by stem cells needs a careful
selection of both stem and glioma cell types.
Aims
By implanting feno- and genotypicaly homogeneous neuroepithelial stem cells, we
intended to investigate the influences of the host brain environment on stem cell
integration. Using in vivo and in vitro models, we intended to find answers to he
following questions:
1.
How the age (developmental stage) of the host does influence the integration of
neural stem cells?
2.
How pathophisiological conditions do influence the fate of implanted stem cells?
3.
Are there conditions, which allow using stem cells as vehicles for delivery of antitumour agents?
Materials and methods

Maintaining of stem and glioma cell cultures

In vitro induction of neuronal differentiation

Chimera-aggregate cultures

BrdU labelling

Cell viability assay (MTT reduction method)

Determination the chromosome number

Cell implantation into adult, newborn and embryonic brains

Cold lesioning of adult mouse cortices

Histological elaboration of brain sections

Immunocytochemistry

Fluorescence microscopy

Statistical analysis
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Results
The continuously proliferating GFP-4C and PLAP-4C cells showed characteristics
that were identical to those of the NE-4C mother clone, and displayed several features
of non-committed neural stem cells. Besides continuous proliferation and nondifferentiated epithel-like morphology, these cells express the stage-specific embryonic
antigen-1 (SSEA-1) characteristic also for mouse embryonic stem cells. The sub-clones
maintained the neurogenic potential of the mother clone as it was shown by their neuron
production upon induction with RA or by the presence of astrocytes prepared from
perinatal brain.
Fate of implanted stem cells in the intact forebrain tissue grafted at different ages
In the intact adult brain, the implanted cells formed compact aggregates during
the first week. After intraventricular injections, the aggregates of implanted cells
attached to the wall of the ventricle or to the choroid plexus and a few labelled cells
invaded a narrow layer of the corpus callosum, closest to the ventricle. In the grey
parenchyma, however, the aggregates sharply delineated from the host tissue. By the
third week of intraventricular transplantation, clusters of cells appeared along the major
fibre tracts, e.g. in the corpus callosum and fimbria-fornix, demonstrating that cells
migrated along the fascicles. Some long, labelled processes were also revealed among
the striatal fibers in the striatum-grafted animals indicating that the host fibres provided
substrate for process-elongation. Implanted cells did not migrate into the grey matter,
and the few cells, those stuck along the trans-cortical injection tract, died. Only a few
cell acquired differentiated morphology and displayed neurofilament- or GFAPimmunorectivity at the edge of the aggregates, and sporadically, also inside the
aggregates. The frequency of differentiated cells was very low and did not exceed the
proportion (<0.1%) of NE-4C neurons formed spontaneously in non-induced cultures.
The size of the aggregates increased during the first 3 postoperative weeks both in
the ventricle and in the striatum, indicating that the cells divided inside the aggregates.
The rate of proliferation, however, was rather low: BrdU labelling was not completely
diluted out even by the 21st post-implantation day. By the 6th week, the total volume of
the grafts decreased sharply. 40-46 days after the implantation, clusters of “foreign”
cells were not found anymore, and the brain structure seemed to be restored completely.
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A few single, labelled cells, however, were still observed in periventricular areas along
the lateral ventricle and in the fimbria. Among these cells, differentiated neuron-like
cells were not revealed.
In neonatal hosts, the grafted cells grew in expanding aggregates, often distorting
the neighbouring host tissue. Enlarged implants survived for a long time: they were
present even after 6 weeks of implantation, and sometimes they resembled huge
tumour-like inclusions in the subcortical regions. Inside the aggregates, some bipolar
cells showed neurofilament- or GFAP-immunoreactivity from the end of the third week.
Morphologically differentiated NE-4C neurons and in-growing neurites, however, were
not found.
In comparison to adult hosts, the neonatal forebrain environment seemed to
stimulate the proliferation of NE-4C progenitors, but did not facilitate their migration,
integration and neural differentiation.
The data obtained from implantations into mouse foetuses reflected the difficulties
of in utero targeting. The applied protocol allowed reliable implantation only between
E13-E16. Moreover, in order to protect the injected and potentially damaged pups, they
were removed from the uteri by caesarean section before birth. Thus, the protocol
allowed only a limited - 3 to 6 days - period of intracerebral development for the grafted
cells. As expected, grafted NE-4C cells did not develop mature neuronal features during
the short survival period. In the foetal forebrain vesicles, the implanted cells attached to
the choroid plexus or intercalated among the host cells in the ventricle wall. They did
not separated from the host cells and their expansion was much slower than that in the
newborn environment.
The data demonstrated that the fate of implanted stem cells was highly determined
by the host environment.
Fate of implanted stem cells in patho-physiologically altered adult brain tissues
NE-4C stem cells expressing histological marker proteins were implanted into the
brain of host animals damaged either by cold lesion or by introducing glioblastomas.
Lesions on adult mice were produced by cryogenic injury method. In this
reproducible cortical injury model, the damaged tissue is characterised by a necrotic
core region and a peri-lesion rim area. To avoid unnecessary loss of implanted cells
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caused by vasogenic brain oedema, neural stem cells were implanted after a 7-day postlesion recovery period.
By the end of the first post-implantation (e.g. second post-lesion) week, GFP-4C
cells were found in all (lesioned or control) implanted brains. By the fourth week,
implanted cells were revealed only in 11% of non-lesioned, while in 61% of lesioned
animals. By the end of the 8th post-implantation week, GFP-4C cells were not found in
any of the intact host brains. Among the lesioned hosts, 69% of the animals carried
viable grafted cells. Inside the GFP-4C clusters enlarging in the lesioned areas, a few
GFAP- and GFP double-positive cells with astroglia-like morphology appeared by the
end of the second post-implantation week. NeuN immunoreactivity – a marker of
neurons -, however, was not detected in GFP-positive cells.
Depending on the lesioned or intact host environment, significant differences were
found in the survival and proliferation of GFP-4C cells. The observed tumour-like
expansion in the lesioned areas led us to retrieve the progeny of grafted cells from the
lesioned host brains after long-term (~ 6 weeks) intracerebral survival. In vitro cell
biological investigations demonstrated that re-cultivated progenies did not differ from
the “mother” stem cell clone. The data showed that the altered “fate” of GFP-4C stem
cells in the peri-lesion environment was an adaptive cellular response to environmental
signals, rather than a manifested shift in the main characteristics of the cell line.
For producing targetable tumour-models, gliomas were to be established in the
forebrains of adult mice. For selecting the appropriate glioma, several glioma-lines (C6,
U87, LL, and Gl261) had been investigated. To find matches between various types of
glioma cells and neural stem cells, rapid in vitro methods have been elaborated.
The proliferation-promoting effects of secreted soluble factors were investigated
by 3H-thymidin incorporation assays on cells maintained in the presence of conditioned
media (CM) derived from other cells in question. CM taken from NE-4C cells increased
the proliferation of astrocytes and C6 cells, but had no effect on the proliferation of U87
and Gl261 cells. 3H-thymidin incorporation by NE-4C cells was not influenced by CM
taken from any glioma-lines.
The co-adhesive behaviour was investigated by producing chimera-aggregates
from different pairs of GFP labelled and non-labelled cells. The C6, U87 and LL cells
were segregated from the aggregates of NE-4C stem cells. Gl261 glioma cells, however,
intermingled readily with NE-4C cells. In this pairing, the different cells were evenly
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distributed throughout the whole aggregate. Interestingly, primary astrocytes could
aggregate with all types of gliomas, but segregated from GFP-4C cells.
On the basis of the above data, Gl261 was selected for in vivo experiments on
tumour targeting by NE-4C stem cells. For tumour targeting two sorts of implantation
experiments were carried out. In the first group of experiments tumours had been
established by injecting Gl261 glioma cells and NE-4C stem cells were implanted three
days later. In other series of experiments, Gl261 and NE-4C cells were mixed and
implanted together into the forebrain of adult mice.
In the first group of experiments on the 7th post-implantation day, PLAP-4C stem
cells were found near to the Gl261 gliomas. By the 14th post-implantation day, however,
PLAP-4C cells were not found in any animals. Migration of stem cells to the tumour
mass was not detected in any animals indicating that PLAP-4C cells were either not
attracted by Gl261 cells, or could not move across the brain parenchyma toward the
tumour. Moreover, the rapid growth of Gl261 cells impaired the survival of PLAP-4C
cells.
Mixed suspensions of Gl261 and PLAP-4C cells produced common, expanding
tumour-inclusions in several regions of the forebrain. On the 7th post-operative day,
PLAP-4C and Gl261 cells were found in all injected animals, and the tumours expanded
in all animals by the second post-implantation week. The distribution of PLAP labelled
and non-labelled cells inside the tumours indicated that PLAP-4C cells proliferated at a
lower rate than Gl261 cells. Inside the tumours, stem cells persisted also in those
forebrain areas - as in the cerebral cortex -, which did not support their survival in the
non-tumourous adult brain. While Gl261 tumour-mass invaded the brain parenchyma,
PLAP-4C cells resided inside the tumour and did not migrate into the intact brain tissue.
PLAP-4C cells did not leave the tumour-mass even at the ventricular wall, in contrast to
their behaviour in the healthy adult forebrain, where they settled in the subventricular
zone. The observation indicated an adhesion preference of NE-4C stem cells for the
Gl261 environment above any regions of the host tissue.
Conclusions
Pheno- and genotypically identical NE-4C neural stem cells were implanted into the
brain tissue in order to approach the question: why inherent neural stem cells have a
limited capacity to replace neurons decaying in neural diseases or brain injuries.
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According to our previous results NE-4C cells migrate for long distances from the
graft in the early embryonic brain. By integrating into the host tissue many of the cells
differentiate into neurons. In the young postnatal brain, the cells proliferated and
survived long (more than 6 weeks) periods, but did not differentiate. Their large-scale
(occasionally tumour-like) expansion calls special attention to use non-committed stem
cells for cell therapies in young, maturating tissues. In the intact adult brain, nondifferentiated neural stem cells survived a maximum of 4 weeks, displayed limited
proliferation, and the rate of differentiations was very low. The adult brain parenchyma
prevented their migration. The data indicated that non-committed proliferating
neuroectodermal progenitors, in their native state, can not be used for replacement of
adult neural tissue elements. Long-term survival was observed only in neurogenic areas of
the adult brain. For long-term therapies the settlement of non-committed progenitors
among inherent neural stem cells might bear some clinical importants.
In cryogenic cortical lesions, the implanted stem cells survived for a long time, but
neural tissue-type differentiation was not detected. The rate of proliferation however was
comparable to that observed in the young postnatal hosts. The data demonstrate, that postlesion area provides “niche” for survival and multiply of non-committed stem cells. As a
future possibility, some – yet unknown – extrinsically applied factors might help to
initiate the tissue type differentiation of stem cells settled in lesioned areas.
In vitro tests elaborated to study the cellular interactions between stem- and tumour
cells indicated that stem cells can increase the rate of proliferation of some but not all
gliomas. In vitro studies helped to select appropriate stem-tumour cell pairs for
experiments on tumour targeting. Implanted NE-4C cells however did not respond to the
assumed chemo-attractant signals provided by Gl261 intracranial tumours. They did not
migrate towards the tumour through the intact brain parenchyma. NE-4C cells, those
developed inside the tumour, displayed a life-span enough to deliver gene-products inside
the tumour. Further developments are needed to elaborate techniques for tumour targeting
and basic research results are necessary to understand the nature of proteins to be deliver.
The data showed that the intracerebral fate of implanted neural stem cells is
determined by the host environment. Further experiments are in progress to determine
the intracerebral fate of in vitro pre-differentiated neural progenitors in various brain
environments.
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Publication list
Demeter K, Herberth B, Duda E, Domonkos A, Jaffredo T, Herman JP, Madarász E.
2004. Fate of cloned embryonic neuroectodermal cells implanted into the adult,
newborn and embryonic forebrain. Exp Neurol. 188(2):254-67.
Demeter K, Zádori A, Ágoston VA, Madarász E. 2005. Studies on the use of NE-4C
embryonic neuroectodermal stem cells for targeting brain tumour. Neurosci Res.
53(3):331-42.
Ágoston VA, Zádori A, Demeter K, Nagy Z, Madarász E. Altered behaviour of neural
stem cells in intact and lesioned brain areas. 2007. Neuropathology and Applied
Neurobiology in press.
Szlávik V, Vág J, Markó K, Demeter K, Madarász E, Oláh I, Zelles T, O’Connell BC,
Varga G. 2007. Matrigel-induced acinar differentiation is followed by apoptosis in
HSG cells. Journal of Cellular Biochemistry in press
Acknowledgements
I thank to Emília Madarász for the opportunity to work in her laboratory and her
professional leading during the common work.
I would like to thank to members of Laboratory of Neuronal Cell and
Developmental Biology. Special thanks to Anita Zádori M.D., Balázs Herberth Ph.D.,
and Viktor Ágoston M.D., in addition Kornélia Barabás, Jonathan Davis, Katalin Gaál,
Nóra Hádinger, Márta Jelitai Ph.D., Zsuzsanna Környei Ph.D., Ferencné Laczkó, Inna
Levkovets Ph.D., Károly Markó, Piroska Nyámándi, Barbara Orsolits, Katalin Schlett
Ph.D., Vanda Szlávik, Krisztián Tárnok, Balázs Varga, Patrícia Varju Ph.D., Erzsébet
Vörös, for their helpful assistance, friendly atmosphere during the common years.
Last but no least I would like to thank to my wife, my parents, and to my friends.
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