RISK-IR: Risk, Stem Cells and Tissue Kinetics-Ionising
Radiation
Workpackage 3 review and assessment report – 27 months
Due date:
09 February 2015
Actual submission date: 19 February 2015
Status:
Final
Document code
RISK-IR-[WP3 internal report]-[all Partners]-[27month]-[19-02-2015]
Lead beneficiary
3, 4, 7, 9
Authors
Michael Rosemann
Contributors
Panagiota Sotiropoulou, Manuel Serrano, Umberto Galderisi, Michael
Rosemann
Approval
Michael Rosemann, 19 February 2015
Background
RISK-IR’s contractual reporting period for EC is 18 months. Internal reporting is at 6 monthly intervals, this
will allow any issues to be identified early and corrections to be made in a timely fashion ahead of EC
reporting. WP leaders and the Coordinator will need information from all beneficiaries in order to follow the
progress of the project and to complete internal and formal periodic reports in time. In each reporting
period WP leaders will prepare a WP Review and Assessment Report, drawing on information included in
partner reports. At the appropriate times the WP level reports will serve as an input for the Periodic report
requested by the EC.
Instructions
This document is the template for the workpackage review and assessment reports. Each WP leader is
requested to prepare one report including information on all its activities by WP and Task based on the
information received from partners.
The completed report should be provided to the Coordinator by the deadline indicated on cover page.
In case you have any questions about the template or information to be included, please contact the coordinator
In the sections and tables below, please include brief text on the following where indicated:
1. A contribution to the publishable summary
2. Objectives
For the workpackage and each Task, please provide an overview of your objectives for the reporting period
in question.
3. Work performed and progress achieved
Please describe briefly the work performed during reporting period and progress achieved with reference to
planned objectives. Highlight clearly significant results if applicable.
4. Deviations and corrective actions
Please report if there are any deviations from the original work plan.
 If applicable, explain the reasons for deviations from Annex I (Description of Work)and their impact
on other tasks as well as on available resources and planning.
 If applicable, explain the reasons for failing to achieve critical objectives and/or not being on
schedule and explain the impact on other tasks as well as on available resources and planning
 Please provide a statement on the use of resources. If applicable, highlight and explain the
deviations between actual and planned person-months for this WP.
 If applicable, propose corrective actions.
5. Please also complete relevant parts of the deliverables and milestones tables for the WP and provide
details of publications etc.
2
WP3 Age dependency, radiation quality dependency and species/tissue
specificity of responses
WP3 objectives
Adult stem cells maintain their identity throughout the lifespan of an organism. Their role in the development of
malignant diseases is poorly understoud, but the fact that under genotoxic stress these cells could accumulate genetic
changes over many years makes them an important subject to study ther response after ionizing irradiation. Under
physiological conditions adult stem cells have a virtually unlimitted proliferation potential and therefore could pass a
mutation to a large number of cell progeny. Adult stem cells from skin, bone marrow and mesenchymal tissues are
currently frequently used in autologous cell therapies, after their ex-vivo expansion. No long-term clinical data are
available yet which address the issue of genetic stability of those cells after re-transplantation. Therefore we can at the
moment only speculate of the influence on these cells of a preceding radiation exposure, accumulation of background
radiation with age, or the degree of ex-vivo growth stimulation. iPS cells, widely believed to provide a universal,
personalized ressource to replace degenerated or diseased organs and tissues, are also a potential carrier and
transmitter of genetic alterations, depending on the cell origin from which they are derived. Here, a preceding
radiation exposure might not only have long-term detrimental effects in terms of increased cancer risk (as shown by the
easy and frequent induction of teratomas from iPS), but also an accute failure of stemcell potency. WP 3 tries to
address the problem of the radiation-effects onto skin-, mesenchymal- and iPS stem cells and an interference with the
cell age. In particular we are interested in the function of genes involved in growth and cell cycle regulation (P53, Rb1,
Rb2, ARF) and differentiation (Ptch) to better understand to what degree these genetic factors govern the long-term
stability and potency of adult stem cells.
WP3 achievements
During the reporting the group at CNIO succesfully generated inducible murine iPS cells with a defect in p53 and
p16/19Arf and could thereby validate that wt p53 reduces iPS reprogramming, wheras wt p16/19Arf promotes this
mechanism. As already demonstrated in the last reporting period, the formation of teratoma can be used as a marker
for iPS totipotency. The iPS/p53 or iPS/P16-P19 double transgenic mice will be exploited next for the measurement of
radiation-induced transformation and senescence (Task 3.2).
Murine MSCs of a Rb1 wt genotype where found to exhibit in-vitro a low-frequency formation of aberrant foci (> 3/106
cells plated). It was found that the frequency of these foci increases after LD gamma irradition in cells of 9 month old
mice more pronounced than in cells of 4 month old mice. It has to be verified that these aberrant clones represent early
stages of a malignant transformation (Task 3.3).
The impact of RB1 deficiency on the radiation-induced aging (loss of stemness) of MSCs was found to differ between
human and murine cells. Whereas human MSCs with knocked-down RB1 show a cell-cycle arrest and increased
senescence, these features seem to be compensated in murine MSCs by the presence of the Rb paralog genes Rb2 and
p107. In line with this finding, we see that intermediate doses of gamma irradiation (1-4 Gy) can induced senescence
only in human, but not murine MSCs, whereas apoptosis is absent in MSCs of both species. It is interesting to note that
albeit senescence is not induced in irradiated murine MSCs, a significant increase in premature adipogenic
differentiation was observed at doses as low as 200 mSv (Task 3.4).
For the transformation of skin stem cells into basal cell carcinoma (BCC), a sensitized K14cre;Ptch+/fl mice was
monitored for up to 1.5 years after gamma irradiation. High dose irradiated mice (5 Gy) as a control presented multiple
invasive BCCs, whereas non-irradiated mice were free of any such lesion. After low dose irradiation (50 mGy), mice
developed a much smaller number of BCCs, but the morphology and progression of the LD induced tumors were
comparable to the BCCs seen after 100 x higher dose. These result show that low dose radiation can induce indeed BCC
in individuals with a genetic predisposition targeted to the skin stem cells, and that this triggers the formation of more
tumors, without changing their molecular or cellular characteristics (Task 3.5).
WP3 expected final results and impact
By exploiting the inducible iPS mouse in combination with a genetic p53 and p16/p19ARF defect we will finally
understand if a LD irradiation of iPS cells in combination with these two defects bears a risk for genetic instability or
malignant transformation. This will have a great impact for safety aspects of any future therapeutic use of human iPS
cells. Adult stem cells such as MSCs and skin stem cells remain present in an organism for its entire life time. Therefore,
they can in theory accumulate low frequent genetic alterations to a much larger extent than commited or differentiated
somatic cells, which have a relatively short turn over. It is of big importance for the projection of radiation risk to
understand, if and to which extent a long term accumulation of genetic lesions induced by LDR irradiation in adult stem
cells contributes to malignant late effects.
3
Task 3.1
Radiation effects on iPS reprogramming and their multipotency
Objectives:
Studies of the influence of normal aging and low dose irradiation on stem cell regulation will be carried out
on iPS cells derived from murine fibroblasts. Using a novel doxycycline-inducible model we intended to
generate a highly versatile system to manipulate stemness, test multipotency of stem-cells in a living mouse
and analyse changes by administering genotoxic stress by ionizing radiation. A succesful generation of a
Dox-inducible iPS mouse shall provide stem cell populations form multiple tissues and at different ages,
thereby facilitating the further investigation at the next stages of the project of the interplay of age and IR
exposure onto stem cell stability and potency.
Work performed and progress achieved:
In the 18 – 27 month project period no further experiments were carried out on task 3.1.
Work will continue on this task in the next project period.
Deviations and corrective actions: None
Task 3.2
p53 / ARF knockdown in irradiated murine iPS
Objectives:
The stress-response genes p53 and Ink4/ARF, known to limit iPS induction, will be conditionally knocked
down in these cells to test if they are modulators of radiation-responses to high and low doses in stem-cells.
This study will investigate the potential risk modulatory role of p53 and Ink4/ARF in irradiated iPS stem cells
in vivo. Earlier work demonstrated that p53 knockdown in iPS cells reduces the DNA damage response due
to telomere-atrition and causes an almost complete absence of early apoptotic cells.
Work performed and progress achieved:
We have analysed the involvement of tumour suppressor genes (TSG) in the reprogramming process in vivo,
using the ”reprogrammable” mouse strain (i4F) previously generated by us (Abad et al., 2013), which
ubiquitously express the four Yamanaka factors in a doxycycline inducible manner. Preliminary results show
that induction of the four Yamanaka factors in vivo upregulates the expression of p53, p16, p19, and p21 in
several tissues, in agreement with the published in vitro data. Moreover, the activation of the
reprogramming process in vivo induces a senescence programme as demonstrated by the induction of IL6
and the detection of Senescence Asociated-ß galactosidase (SA-ßGal) positive cells in the induced i4F mice
(Fig. 1).
Figure 1. (a) Upregulation of TSG and IL6 expression levels measured by qRT-PCR in the kidney of i4F mice (black) compared to WT
mice (grey) treated with DOX 1mg/ml for 1 week (b). IHC against Nanog (pink) and p21 (brown, left) or p53 (brown, right) shows
coexpression of these markers in the stomach of induced i4F mice c. SA-ßGal positive cells in the pancreas of i4F mice treated with
DOX 1 mg/ml for 1 week.
To further characterise the interplay between reprogramming and senescence in vivo, and the involvement
of TSG in in vivo reprogramming processes, we have generated reprogrammable mouse strains deficient for
p53 and Ink4aArf by crossing our ”reprogrammable” mouse strain with mouse strains deficient for p53 and
Ink4aArf. Both tumour suppressors are important cell-autonomous barriers to reprogramming, as well as,
important mediators of senescence in those cells that fail to be reprogrammed. Interestingly, we have
observed opposite effects with these tumour suppressors: in the case of a p53-null background, in vivo
reprogramming (measured by latency and number of teratomas) is accelerated; whereas, in the case of a
p16/p19Arf-null background, in vivo reprogramming is delayed. This correlates with the initial proliferative
4
response elicited by the 4 Yamanaka factors (Fig. 2).
Figure 2. Pancreas of the reprogrammable mouse strains treated with DOX 0.2 mg/ml (5 days) and stained with anti-BrdU.
iPS cells of the different TSG-deficient genetic backgrounds can now be isolated and subjected to the planed
gamma-irradiation studies.
Deviations and corrective actions: none
Task 3.3
in-vitro transformation of murine MSCs after IR
Objectives:
Murine MSCs are known to undergo spontaneously a low-frequency transformation during in vitro growth.
We will test by long-term monitoring the extent to which this malignant transformation is promoted after
sub-lethal radiation-exposure to alpha and gamma radiation at low doses. To understand to what extend invitro transfomed MSCs are of relevance for radiation-induced carcinogenesis, we aim to investigate the
influence of cell age (age of donor mouse) and the potential promoting influence of a pre-existing defect in
the Rb1 tumor suppressor gene.
Work performed and progress achieved:
Using hypoxic in-vitro growth conditions we are able now to maintain murine MSCs in culture for ~ 6 weeks.
During this period, 20 – 30 colony forming units per mouse femure expand to 1-2 million cells. The number
of MSC colonies observed after plating was not significantly reduced by in-vivo gamma-irradiation with
50mSv or 150mSv, nor did we observed a clear reduction in clonogenic survival in-vitro in the dose range
between 0 and 200 mSv.
We now quantified the appearance of aberrant foci of MSCs, which exhibit a piled-up growth pattern,
focal loss of cell-adhesion to the plastic surface and aggregation of round shaped cells (Fig. 3).
100 µm
No.
aberrant
foci
15
10
4m
5
9m
0
control
A
B
50mGy
150mGy
C
Fig 3: Aberrant clones in murine MSCs culture. 4x dark field microscopy (A), 10x phase contrast (B) with 100µm scale bar, number of
aberrant foci counted in MSC cultures from 4 month (blue) and 9 month (red) old donor mice, after in-vitro gamma-irradition with
0, 50, or 150mSv.
The first data suggest that in MSCs from 9 month old mice, but not from 4 month old mice, 150mSv gammairradiation can induce a 3fold rise in the number of aberrant foci. To evaluate the influence of an Rb1 defect
on this phenomenon, we are currently set up Rb1+/- MSCs and MSCs with a lentiviral-induced -/- genotype.
A recent study in human MSCs described the formation of such aberrant foci after treatment of the cells
with the chemical carcinogen 3-MCA. In Tang et al (2013, PlosONE) it was shown that these foci are
5
tumorigenic in nude mice. We will therefore collect the radiation-associated MSC foci and test them in the
next step for tumorigeneity after transplantation.
Deviations and corrective actions:
The planned alpha-irradiation of murine MSCs to investigate alpha-induced genomic-instability and
transformation could not be done yet, due to the decommissioning of the source at the Helmholtz-Center
Munich. We are currently negotiating the technical requirements for an alpha-irradiation at the RISK-IR
partner institution 02 (CEA/FR). A second option would be to replace alpha-irradiation with fission-neutrons
from the Munich University research reactor (neutrons yield a similar RBE as the originally planned alpha
particles).
Task 3.4
in-vitro aging of human and murine MSCs after IR
Objectives:
In both murine and human MSCs we will follow the natural aging process and its potential acceleration by
irradiation at low and high dose, using assays such as telomeric attrition, senescence in the irradiated cells
and loss of chromosomal integrity. Since MSCs from both species are available with different Rb1-status in
the partner labs, the influence of this a functional Rb1-pathway on irradiated MSCs will be analyzed.
Work performed and progress achieved:
IR effects on human bone marrow Mesenchymal Stem Cells (MSC)
Human MSC were irradiated with 40 and 2,000 mGy gamma rays. Cells were allowed to recover and DNA
damage was evaluated 1, MSC exposed to low radiation did not show any radio-resistance phenomenon as
described for other components of bone marrows, such hematopoietic stem cells and fibroblasts. This
suggests that they may be one of the more sensitive components of bone marrow.
The main consequence of low radiation exposure, besides a temporal drop of cell cycling, is the trigger of
senescence (increasing between 40 and 2000 mGy), while the contribution of apoptosis is marginal (this was
further confirmed at supraletal doses up to 20 Gy). Senescence is clearly associated with loss of stemness, in
particular leading to a reduced clonogeneity of MSCs.
Increase in ATM staining one and six hours post irradiation and its drop to basal level at 48 hours along with
an enduring gamma-H2AX staining suggested that MSC activated properly the DNA repair signaling system
but some damages persisted unrepaired. At the opposite gamma-H2AX can remain bound to unrepaired
DNA, as suggested by the kinetics analysis of gamma-H2AX clearance after IR or other DNA damaging
agents. The existence of persistent unrepaired DNA foci may be the trigger of senescence phenomena as
already evidenced by Campisi’s team, who evidenced that persistent foci of damaged DNA, termed DNASCARS sustain damage-induced senescence growth arrest.
Endurance of gamma-H2AX foci is mainly observed in non-cycling cells (Ki67-) indicating that the impaired
DNA repair capacity of irradiated MSC seemed mainly related to reduced activity of NHEJ system rather
than HR. Indeed, the NHEJ is the only double strand breaks repair system that is active in non-cycling cells.
Data on the activation of NHEJ and HR (DNA-PK and RAD51 immunostaining) further strengthen our
hypothesis.
In conclusion our data suggest that human bone marrow MSC are sensitive to very low radiations and
trigger senescence due to impaired autophagy and DNA repair capacity. The presence of senescence rather
than apoptosis may be more dangerous for the proper functioning of bone marrow.
IR effects on mouse bone marrow Mesenchymal Stem Cells (MSC)
We are performing the same experiments we carried out on human MSCs also on murine MSC and
preliminary data appear to overlap those observed in human cultures.
Silencing of RB1 gene on human and mouse MSC
Both in human and in murine MSCs we have silenced the RB1 gene (using lentiviral expression of shRNAs
and/or siRNAs) to compare the effect onto the entire cellular pathway in both species. Human MSC with
silenced RB1 showed reduced proliferation, accumulation in G1 phase and increase in senescence while in
mouse MSC with silenced RB1 we did not observe significant changes in cell cycle profile and senescence
6
compared with wild type cells. These differences seem to ascribed to partial compensation by the RB1
paralog genes RB2/P130 and P107. First results from gamma-irradiated, human and mouse MSC with
silenced RB1 suggest that for the canonical RB pathway (cell-cycle regulation, differentiation) a more
complex interaction of the tree retinoblastoma paralogs takes place in mice. It remains to be tested if for
the endpoint of chromosomal stability, an Rb1 defect alone is sufficient to causes an impairment, or if a
compensation by Rb2 and P107 rescues the defect.
In long-term cultures of irradiated murine MSCs the effect of premature loss of stemness (aging) was
measured using senescence, apoptosis, premature differentiation and impairment in a competitive growth
assay as endpoints.
Using pan-Caspase IF, a basal level of ~3% apoptotic cells were always present in the murine MSC culture.
SA-ßGal staining detected < 0.5% senescent cells in the murine MSCs during 6 weeks growth. Neither for
apoptosis nor for senescence, a significant increase was found after irradiation with 50mSv, 200mSv, 1Sv,
2Sv and 4Sv (observation up to 48 hours p.i). This clearly shows that murine MSC are relatively resistant for
radiation-induced apoptosis and senescence.
We found, however, that after irradiation with 200mSv (suggestive) and 500mSv (significant) murine MSCs
undergo spontaneous, premature differentiation into adipocytes (Fig 4).
A
B
Fig 4: Adipogenic differentiation (by oil-red staining) in murine MSCs 48 h after gamma-irradiation with 500mSv (A). Area of cells
exhibiting adipogenic differentiation (6 well plate) following gamma doses from 0.1mSv to 4Sv (green:hypoxia, blue:
Normoxia). Significant increases in % adipogenic differentiation relative to control: p<0.05 (500 mSv) p<0.01 (2 Sv),
Differenc between hypoxic and normoxic conditions not significant.
This implies that MSCs can lose their three-potency and stop self-renewal as a low-dose radiation effect.
This is a clear aging effect, considering that an increased adipogenic differentation reduces the pool of MSCs
available for the regeneration of connective, osteogenic or chondrogenic tissue.
Deviations and corrective actions: None
7
Task 3.5
Radiation-induced changes in murine epithelial stem cells and skin cancer induction
Objectives:
Detection of potential malignant transformation of the distinct types of skin epidermal stem cells, and how
this potential is modified in predisposed and aged individuals.
Work performed and progress achieved:
Tumorigenic potential of low dose irradiation in predisposed individuals
Distinct mutations have been shown to predispose individuals to specific types of cancer, such as Brca1 and
Brca2 mutations in breast and ovarian cancer and KRas mutations in skin cancer. However, these mutations
have to be supplemented by other gene alterations, in order to generate tumours, and thus it could be
possible that these individuals should avoid exposure to low dose radiation, should this proves to induce
higher or earlier incidence of tumour formation. To investigate the effect of low dose radiation in the
distinct types of epidermal SCs in cancer-prone individuals, we used mice were we deleted the one allele of
the Hedgehog inhibitor Patched [often mutated in human basal cell carcinoma (BCC), the most common
type of skin cancer] specifically in the skin epidermis (K14Cre;Ptchfl/+). Patched heterozygous (Ptch+/-) mice
develop skin tumours that recapitulate human BCC around 1 year after a single dose of ionizing radiation (34 Gy).
We have previously shown that upon 50 mGy total body irradiation, bulge SCs do not undergo apoptosis
neither in WT, nor in K14Cre;Ptch+/fl mice, while SG SCs commit apoptosis only in WT mice upon 50 mGy
irradiation, raising the question whether the surviving SCs of either type could exhibit chromosomal
alterations potentially leading to cancer development.
To investigate the incidence and timing of cancer development in predisposed mice receiving low dose
radiation, we administered 50 mGy radiation to K14Cre;Ptch+/fl animals. One year following irradiation
mice were sacrificed and tail epidermis sections were investigated for tumour development. The epidermis
of non-irradiated control K14cre;Ptch+/fl mice did not exhibit any lesions, even at 1.5 years of age. As
expected, mice irradiated with 5 Gy, used as positive controls for cancer development, presented multiple
invasive BCCs, and very few dysplastic lesions. Mice irradiated with 50 mGy presented BCCs, at comparable
stage and size to the tumours of 5 Gy-irradiated mice, albeit at much lower numbers (Fig. 5B). This result
shows that low dose radiation can induce indeed BCC in predisposed individuals, albeit tumour incidence is
lower compared to high radiation doses. As expected, the progression of tumours is comparable in both
conditions.
Figure 5: Low dose IR results in cancer development in Ptch heterozygous mice. A. Representative immunofluorescent image of a
basal cell carcinoma in tail epidermis of a K14Cre;Ptch+/fl mouse. B. Number of tumours per cm of epidermis in tail skin of
K14Cre;Ptch+/fl mice without or 1 year after receiving 5 Gy or 50 mGy radiation. Note that although the tumour incidence is much
lower than in high radiation doses, low dose radiation indeed causes cancer in predisposed individuals.
The second most common type of skin cancer is squamous cell carcinoma (SCC). Mutations in the Ras genes
are very common in human SCC, and transgenic mouse models expressing a constitutively active form of
KRas, together with loss-of-function of p53 recapitulate human SCC. To this end, we used
Lgr5CreER;KRasG12D;p53f/f;RosaYFP mice, which develop multiple skin SCCs upon tamoxifen
administration. To date we have analyzed the skin of only 3 mice, all induced with tamoxifen, among which
the 1 was irradiated at 50 mGy, 3 days after the last tamoxifen induction. We monitored the incidence and
8
latency of the tumours, as well as their size and malignant progression. Our preliminary data suggest that
tumour initiation is enhanced in irradiated mice, as shown above in the BCC model, while tumour latency,
size and malignant progression do not appear to be affected (Table 1). As noted above, these results were
obtained from only a small number of animals. More mice are currently under treatment and definitive
results will be presented in the next report.
Table 1: Low dose IR induces squamous cell carcinoma development in KRas transgenic mice.
SCCs/mouse time
for
SCC mean SCC size
appearance
(mm3)
(weeks)
Control 1
3
8
195
Control 2
7
11
107
50 mGy 1 9
11
113
Deviations and corrective actions: None
9
Deliverables
Del.
no.
Deliverable name
WP no.
Lead
beneficiary
Delivery date
from Annex I
(proj month)
Actual /
Forecast
delivery
date
Delivered?
Comments
Yes/No
Dd/mm/
yyyy
D3.1
D3.2
D3.3
D3.4
D3.5
D3.6
D3.7
Expression profiles of
derived iPS cells
(control, after LD
gamma and alphairradiation)
Murine normal and
in-vitro
IR
transformed
mesenchymal stem
cell lines (on Rb1-/-,
Rb1+/- and Rb1+/+
Data on age-related
and
radiationassociated changes
in human and murine
MSC
multipotency,
senescen
Tissues of IR induced
skin cancer in Ptch+/, tg KRAS and
tgKRAS/P53fl-fl mice
Histological
sections of stem
cell regulation in
irradiated skin of
Ptch+/-, tg KRAS
and tgKRAS/P53f
mRNA expression
profiles
(control
and
post
irradiation)
of
murine HF bulge
stem
cells
and
sebaceous
Review paper: Lowdose exposure and
age-dependent loss
of stem-cell potential
31/10/2
016
Pending
30/04/2015
(30 months)
30/04/2
015
Pending
18.0
31/10/2016
(48 months)
31/10/2
016
Pending
Partner
09
(ULBBE)
Partner
09
(ULBBE)
9.0
30/04/2016
(42 months)
30/04/2
016
Pending
6.0
31/10/2014
(24 months)
31/10/2
014
Pending
Partner
09
(ULBBE)
9.0
31/10/2015
(36 months)
31/10/2
015
Pending
Partner
04
(HMGU
-DE)
3.0
30/04/2017
(54 months)
30/04/2
017
Pending
Partner
04
(HMGU
-DE)
18.0
Partner
04
(HMGU
-DE)
18.0
Partner
04
(HMGU
-DE)
31/10/2016
(48 months)
10
Milestones
TABLE 2. MILESTONES
Milestone
no.
Milestone
name
Work
package
Lead
no
beneficiary
Delivery
Achieved
Actual /
date from
Yes/No
Forecast
Annex I
achievement
dd/mm/yyyy
date
dd/mm/yyyy
Comments
MS 13
P53
and
Ink4/ARF
knockdown
WP3
Partner 07
(CNIO-ES)
24
YES
Characteristic
changes in gene
expression profile
MS 14
Low-dose
radiationinduced
changes in iPS
multipotency
and stem-cell
potential
WP3
Partner 07
(CNIO-ES)
36
pending
Whole
genome
mRNA profile and
validation
by
single gene assay
MS 15
In-vitro
transformation
of
murine
MSCs
after
irradiation
WP3
Partner 04
(HMGUDE)
30
pending
Tumorigeneity
after
transplantation
in
kongenic
mice
MS 16
Rb1 influence
onto radiationinduced
transformation
WP3
Partner 04
(HMGUDE)
42
pending
Test
for
epigenetic
changes
and
telomer atrition in
Rb1 wt and +/MSCs and in
transfromed
clones
MS 17
Human
MSC
with
Rb1
knockdown
WP3
Partner 03
(2UNINAPIT)
24
YES
Knockdown cell
lines made and in
use
MS 18
Panel
epigenetic
assays
of
WP3
Partner 04
(HMGUDE)
42
pending
Stable,
reproducible
results
using
either WB, IF or
flow-cytometry.
MS 19
Monitor aging
in LD irradiated
(gamma
and
alpha) human
and
murine
MSC's
WP3
Partner 03
(2UNINAPIT)
42
pending
Reproducible
changes
in
telomere stability,
epigenetic
changes
and
MSC
potency
during aging and
after IR
MS 20
Radiationinduced
(40mGy - 2Gy)
skin
cancer
tissues from wt,
tg KRAS. Tg
KRAS / P53+/and ptch +/mice
WP3
Partner 09
(ULB-BE)
24
YES
Histolopathological
validation
tumer type
of
11
MS 21
Generation of
epithelial
stem-cells
from HB and
SG
WP3
Partner 09
(ULB-BE)
32
pending
Cell
lines
express
typical stemcell
markers
and
mRNA
profile
MS 22
Changes
of
epithelial stemcells
in-vivo
after irradiation
and
characterisation
of
premalignant les
WP3
Partner 09
(ULB-BE)
40
pending
Similarity of LD
radiation
induced
changes in invitro epithelial
stem-cells and
in in-vivo skin
stem cells
12
Dissemination activities
a) Scientific peer-reviewed publications
Please list here any peer-reviewed scientific papers that were published in the reporting period
b) Other dissemination activities
Please list here any other publications/reports/posters that were published in the reporting period.
Staff working on the project
Please list here the scientific staff working on the project, indicating if PhD student, post-doc, senior
researcher etc and provide email contact details.
Thank you for completing this report!
Send this report to the RISK-IR project office when complete.
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