Lectures L5.1 L5.2 Session 5. Stem Cells and Regenerative Medicine

Transkrypt

Session 5. Stem Cells and Regenerative Medicine
Lectures
L5.2
L5.1
Epigenetic mechanisms of human
induced pluripotent stem cells (iPS)
Cytoplasmic dynamics as a new
marker of embryonic quality Maciej Wiznerowicz1,2
Anna Ajduk
Department of Embryology, Institute of Zoology, University of Warsaw,
Warsaw, Poland
e-mail: Anna Ajduk <[email protected]>
Quality of an oocyte’s cytoplasm is an important factor affecting pre- and postimplantation development of the future embryo. It can be compromised for example by aging,
both maternal (i.e. related to the female's age) and postovulatory (i.e. related to the time between ovulation and fertilization). Recently, we have establish a new non-invasive
method to assess cytoplasmic quality of fertilized oocytes.
It involves a Particle Image Velocimetry (PIV)-based analysis of cytoplasmic movements triggered by a fertilizing
sperm. PIV method is commonly used in fluid dynamics to
follow movements of amorphous objects. We have found
that cytoplasmic movements depend on Ca2+ oscillations
that are also induced by fertilization. Each time Ca2+ level
increases, a contraction of the actomyosin occurs and this
in turn induces a directional cytoplasmic flow. Therefore,
analysis of cytoplasmic flows provides unique information
about functionality of Ca2+ siganlling and cytoskeleton in
the oocytes. In experiments performed on a mouse model,
we have shown that a specific temporal pattern of the cytoplasmic movements (very frequent fast twitches with very
slow movements in the intervening periods) correlates with
a low developmental potential of the embryos. Currently,
our method of embryo assessment is tested in human embryos in a clinical setting.
1Gene Therapy Laboratory, Department of Cancer Immunology,
Poznan University of Medical Sciences, Chair of Medical Biotechnology,
Poznań, Poland; 2Gene Therapy Laboratory, Department of Cancer
Immunology, Greater Poland Cancer Centre, Poznań, Poland
e-mail: Maciej Wiznerowicz <[email protected]>
Induced pluripotent stem cells (iPS) cells are generated by
dedifferentiation of adult cells through the forced expression of few embryonic transcription factors. The reprogramming process involves ordered and global epigenetic
alterations including histone modifications and DNA methylation that progressively silence expression of lineage-specific genes whilst enabling transcription of embryonic factors genes. We explored the role of KRAB-containing zinc
finger proteins (KRAB-ZFPs) and their cofactor TRIM28/
KAP1 during the reprogramming process. The results of
our work strongly suggest that knockdown of TRIM28/
KAP1 expression facilitate de-differentiation of human
primary fibroblasts towards the iPS cells. In the other hand
the inhibition of TRIM28/KAP1 function in established
human pluripotent cells results in progressive loss of their
self-renewal potential in contrast the the wild-type human
iPS cells. Additionally, our parallel lines of research clearly
demonstrated that binding of TRIM28/KAP1 through
KRAB-containing transcriptional repressors results in
specific methylation of the cellular promoters. Finally, we
have revealed that epigenetic factors recruited by TRIM28/
KAP1 controls retrotransposition events during the reprogramming of human somatic cells to pluripotency. Taken
together, our results demonstrated novel role for TRIM28/
KAP1 protein in the reprogramming process and self-renewal of human iPS cell as well as their involvement in
modeling landscape of genomic DNA methylation during
that process. In-depth understanding of epigenetic mechanisms involved in the cellular reprogramming and self-renewal of human iPS cells will have implications for basic
research and may pave ways to novel therapies.
Toruń, September 2nd–5th 2013
74
L5.3
L5.4
Tissue regeneration in FOXN1 deficient
(nude) mice: isolation and analysis of
ear mesenchymal stem cells (EMSC)
Identification of the mechanisms
regulating differentiation of stem/
progenitor cells during regeneration of
the injured central nervous system
Barbara Gawronska-Kozak
Institute of Animal Reproduction and Food Research of Polish Academy
of Sciences, Olsztyn, Poland
e-mail: Barbara Gawronska-Kozak <[email protected]>
Regeneration, as opposed to wound healing, is characterized by lack of scarring, reconstruction of normal tissue
architecture and regrowth of damaged tissues. The phenomenon of regeneration has been almost completely lost
in mammals whose damaged tissues undergo wound healing processes characterized by scar formation. However,
mice carrying a mutation that inactivates transcriptional
factor FOXN1 (nude mice) display an unusual ability for
regeneration, that is, scar-free skin wounds healing and
tissue regeneration in holes punched into their external
ears. Cells isolated from external ears of regenerative and
non-regenerative strains of mice display features of mesenchymal stem cells, including: self-renewal, clonogenic
potentials, differentiation into adipo-, osteo-, chondro- and
myolineages, expression of stem cells factors: (Oct3/4,
Sca-1, CD117) and the ability to form iPSC under stimulative conditions. The data suggest that EMSC (ear mesenchymal stem cells) are capable to support regeneration
in punched ears of nude mice, but their presence is not
sufficient to meet regenerative healing in other strains of
mice. Whether the inactive transcriptional factor FOXN1,
lack of thymus or lack of T-lymphocytes in nude mice create the permissive conditions for regeneration is currently
being investigated. This model of regeneration promises
to provide insights into endogenous dormant stem cells in
future regenerative approaches. Malgorzata Zawadzka
Nencki Institute of Experimental Biology, Department of Cell Biology,
Warsaw, Poland
e-mail: Małgorzata Zawadzka <[email protected]>
Demyelination is a pathological process that occurs in the
central nervous system (CNS) when myelin sheaths responsible for the integrity of axons and guidance of signal are
destroyed. Natural, endogenous response to demyelination
is remyelination, a regenerative process driven by the progenitor cells — the precursors of oligodendrocytes (OPC).
We have shown that during the regeneration of injured
CNS white matter both processes, remyelination and the
reconstruction of blood vessels, take place. Moreover, depending on the microenvironmental clues in injured tissues, oligodendrocyte precursor cells can differentiate on
a classic pathway giving rise to oligodendrocytes or on
an alternative pathway to Schwann cells and astrocytes.
Alternative pathway selection is closely related to the settling of the precursors within the vascular niche, which
substantially differs functionally from tissue that does not
contain blood vessels. With the precise technique of laser
capture microdissection we separated vascular niche from
niche without blood vessels to investigate changes in global
gene expression using the cDNA microarrays. Based on
comparative bioinformatic analysis we selected candidate
genes which significantly discriminated both niches. These
analysis revealed genes involved in differentiation as well
as genes involved in angiogenesis and neovascularization.
Our results indicate that OPC differentiation is regulated
by several signal transduction pathways (BMP, WNT, HH).
In the long-term perspective, we expect that modulation of
signals derived from the vascular environment may be crucial for the development of therapeutic strategies targeting
both enhancement of endogenous remyelination and tissue regeneration after transplantation of exogenous cells.
48th Congress of the Polish Biochemical Society
Session 5. Stem Cells and Regenerative Medicine75
Oral presentations
O5.2
O5.1
Embryonic stem cell-derived microvesicles
as effective carriers of mRNA to target cells
The role of HO-1 and Nrf2 in the
reprogramming of somatic cells to
induced pluripotent stem cells
Marta Adamiak, Sylwia Bobis-Wozowicz,
Zbigniew Madeja, Ewa Zuba-Surma
Department of Cell Biology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Kraków, Poland
Jacek Stępniewski, Tomasz Pacholczak,
Alicja Józkowicz, Józef Dulak
e-mail: Marta Adamiak <[email protected]>
Department of Medical Biotechnology, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland
e-mail: Jacek Stępniewski <[email protected]>
Reprogramming of somatic cells which leads to the generation of induced pluripotent stem cells (iPSCs) is a complex
process requiring many modifications on the level of gene
expression profile, metabolism and chromatin structure.
However, the initial response to the viral vectors delivering reprogramming transcription factors (Oct4, Sox2, Klf4
and c-Myc) involves the elevated production of reactive
oxygene species (ROS) which leads to DNA damage and
p53 activation [1]. Taking this into consideration, the aim
of this study was to evaluate the role of two antioxidative
factors: Nrf2 and heme oxygenase-1 (HO-1) in reprogramming. To induce this process, we produced STEMCCA
lentiviral vectors [2] and transduced primary mouse fibroblasts isolated from tails of Nrf2+/+ and Nrf2-/- as well as
HO-1+/+ and HO-1-/- animals. High efficiency of transduction (reaching 96%), confirmed with GFP-delivering
control vectors, enabled us to optimize the reprogramming
method and to count generated iPSCs colonies in each experiment. It appeared that the lack of Nrf2 significantly
decreased the efficiency of reprogramming even though
the Nrf2-/- fibroblasts proliferated faster than Nrf2+/+.
Additionally, we could observe the elevated production of
reactive oxygen species 48h after transduction of cells with
lentiviral vectors with higher level of ROS in Nrf2-/- fibroblasts. Interestingly, the lack of HO-1 did not appear
to have such strong effect on reprogramming. However,
stimulation of cells with CoPPIX, a potent inducer of
HO-1 expression during initial 7 days of reprogramming
increased the number of generated iPS colonies which indicates that heme oxygenase-1 can play a positive role in
this process. Accordingly, we also observed that the level of
HO-1 was increasing in the later stages of reprogramming.
To confirm pluripotency of generated iPSCs, we picked
and further cultured several colonies of each genotype.
All cells were stably growing for many passages, expressed
markers of pluripotency (like Oct4, Sox2, Nanog, SSEA1,
alkaline phosphatase) and could be differentiated in vitro via
embryoid bodies to the cells originating from three different germ layers. Additionally, for some of the iPSCs, their
differentiation potential was also confirmed in vivo in teratoma formation assay. Importantly, preliminary data show
that HO-1 affect the differentiation of iPS cells since no
teratomas were generated from HO-1-/- cells. These results indicate that antioxidative factors, like Nrf2 and HO-1
play important role in the reprogramming process. Further
studies will help to decipher the exact mechanisms with
which they can influence the generation of induced pluripotent stem cells.
Microvesicles (MVs) are membrane-enclosed cell fragments shed into the extracellular environment by direct
budding from the cell plasma membrane or derived from
the endosomal compartment [1]. MVs are secreted by normal healthy as well as tumor and apoptotic cells and may
contain vast bioactive components such as mRNAs, miRNAs, proteins and lipids. MVs originated from a given cell
type may act as mediators of cell-to-cell communication by
transferring their bioactive contents from parent cells to
cells of other origins [2, 3].
Here, we studied transfer of selected mRNAs by MVs derived from mouse embryonic stem cell line ES-D3 (ESMVs;
isolated by ultracentrifugation) to fibroblastic cell line 3T3L1. We compared the level of transcripts related to pluripotency (Oct4, Nanog, Rex1), cardiomyogenesis (Gata4)
and cell proliferation (Fgf5, H19, Rasgrf1) in ESMVs and
ESCs by real-time RT-PCR method and further visualized
reaction products on agarose gel. The results showed that
ESMVs are enriched in several mRNAs compared to their
parental cell line, including transcripts for Gata4 and Rasgrf1. Surprisingly, expression levels for pluripotency genes,
including Nanog, Oct4 and Rex1 were significantly lower in
MVs. Furthermore, we evaluated the expression of selected surface antigens by cytofluorometric analysis of ESMVs
and revealed the presence of molecules expressed on the
surface of ESCs, such as SSEA-1, Sca-1 and CD105. Lastly,
we investigated the transfer of biologically active content
of ESMVs by incubation of ESMVs with 3T3-L1 cell line
and subsequent analysis of gene expression levels in target
cells. The transfer assay demonstrated that MVs can mediate the transfer of mRNAs from ES-donor cells to 3T3-L1
recipient cells.
These findings suggest that ESMVs and most likely MVs
derived from other early stem/progenitor cell types, may
be important mediators of signaling within stem cell niches and may be potentially utilized as therapeutic tools for
transferring bioactive, regulatory components to target
cells.
References:
1. Camussi G et al (2011) Am J Cancer Res 1: 98–110.
2. Yuan A et al (2009) PLoS ONE 4: e4722.
3. Ratajczak M et al (2006) Leukemia 20: 847–856.
References:
1. Mah N et al (2011) Molecular insights into reprogramming-initiation
events mediated by the OSKM gene regulatory network. PLoS One 6:
e24351.
2. Sommer CA et al (2009) Induced pluripotent stem cell generation using
a single lentiviral stem cell cassette. Stem Cells 27: 543–549. Toruń, September 2nd–5th 2013
76
O5.3
O5.4
Genome-wide DNA methylation
profiles of the regenerating mouse
Trace number of c-Kit+ progenitor cells
is present in explanted human hearts
Bartosz K. Górnikiewicz1, Justyna Podolak1,
Anna Ronowicz2, Piotr Madanecki2, Anna
Stanisławska-Sachadyn3, Paweł Sachadyn1
J. Czapla1, S. Matuszczak1, M. Jarosz1, E.
Wiśniewska2, T. Cichoń1, R. Smolarczyk1, M.
O. Zembala2, M. Zembala2, S. Szala1
1Gdańsk University of Technology, Microbiology Department, Gdańsk,
Poland; 2Medical University of Gdańsk, Department of Biology and
Pharmaceutical Botany, Gdańsk, Poland; 3Department of Biology and
Genetics,Medical University of Gdańsk, Gdańsk, Poland
1Center for Translational Research and Molecular Biology of Cancer,
Maria Skłodowska-Curie Memorial Cancer Cancer and Institute of
Oncology, Gliwice Branch, Gliwice, Poland; 2Deparatment of Cardiac
Surgery and Transplantology Silesian Center for Heart Diseases, Zabrze,
Poland
e-mail: Bartosz Górnikiewicz <[email protected]>
e-mail: Justyna Czapla <[email protected]>
The MRL/MpJ (MRL) mouse possesses remarkable regeneration capabilities that were first observed in the process
of scarless closure of punches in the ear auricle [1]. What
is important, a complete restoration of the excised fragment occurs in the MRL, showing no differences in the
tissues architecture. This process is driven by the blastemalike structure, what resembles epimorphic regeneration observed in amphibians [2]. Since this discovery, the increased
regeneration abilities have been reported in various tissues
and organs including heart, spinal cord, eye retina and cornea and articular cartilage, as well as, they have been connected with abnormal cell cycle [3] and retention of many
features of embryonic metabolism in the MRL mouse [4].
The regeneration depends on re-differentiation of the
new tissues in the damaged area. Therefore the epigenetic
mechanisms seem to be crucial in this process, since they
play an important role in establishing cell identity. In our
research we assume that the re-differentiation of the cells
during regeneration process in the MRL may occur due to
different DNA methylation regulation of genes involved in
development and morphogenesis.
In order to investigate the possible role of this epigenetic
basis in the regeneration phenomenon we have performed
global DNA methylation profiling of heart, liver and spleen
tissues in the adult MRL mouse and standard non-regenerative control strain C57BL/6J (B6). By comparison of obtained DNA methylation profiles we have identified hundreds of differentially methylated genes between these two
strains. The functional analysis of the groups of differentially methylated genes by the DAVID tool (The Database
for Annotation, Visualization and Integrated Discovery)
showed significant increase of genes involved in transcription regulation and embryonic development among the
genes hypomethylated in the MRL mouse. Further studies are necessary in order to examine the potential role of
the identified differences in the genomic DNA methylation
profiles in the regeneration phenomenon.
According to literature data, self-renewing, multipotent and
clonogenic cardiac progenitor cells (CPC, c-Kit+) are located within human myocardium. The aim of this study was
to isolate and characterize c-Kit+ progenitor cells from explanted human hearts. Experimental material was obtained
from 19 adult patients and 7 children. Successful isolation
and culture was achieved for 95 samples (84.1%), derived
from 5 parts of the heart: right and left ventricle, atrium,
intraventricular septum and apex. In our study, an average
percentage of c-Kit+ cells, as assessed by FACS, ranged between 0.7 and 0.9%. The differences in the numbers of
c-Kit+ cells between disease-specific groups, parts of the
heart or sexes were not statistically significant. Nevertheless, c-Kit+ cells were present in significant numbers (1225%) in samples taken from 3 explanted pediatric hearts.
c-Kit+ cells were also positive for CD105 and a majority of
them were positive for CD31 and CD34 (83.7% ±8.6% and
77.8% ±10%, respectively). Immunohistochemical analysis
of heart tissue revealed that most of the cells possessing
c-Kit antigen were also positive for tryptase, a specific mast
cell marker. However, flow cytometry analysis revealed that
cultured c-Kit+ cells are negative for hematopoietic marker
CD45 and mast cell marker CD33. Our findings revealed
trace quantity of c-Kit+ cells in the failing human heart.
Difficulties in obtaining and harvesting large amounts of
c-Kit+ cultured cells may prevent successful autologous
therapy.
Acknowledgements:
The study has been co-financed by European Regional Development
Fund within the framework of Innovative Economy Operational Program (axis: Investments in Innovative Undertakings).
Acknowledgment:
This research has been founded by the NCN grant (2011/01/B/
NZ2/05352).
References:
1. Clark LD et al (1998) Clin Immunol Immunopathol 88: 35–45.
2. Gourevitch D et al (2003) Dev Dyn 226: 377–387.
3. Bedelbaeva K et al (2010) Proc Natl Acad Sci USA 107: 5845–5850.
4. Naviaux RK et al (2009) Mol Genet Metab 3: 133–144.
Posters
P5.2
P5.1
Human and murine very small
embryonic-like cells lack Oct-4A
expression and stemness potential
TRIM28/KAP1 protein controls self-renewal
of human induced pluripotent stem cells.
Wojciech Barczak1, Katarzyna Kulcenty1,
Maciej Wiznerowicz1,2
e-mail: Wojciech Barczak <[email protected]>
Human induced pluripotent stem cells (hiPS) are generated by epigenetic reprogramming of somatic cells through
the exogenous expression of four transcription factors
(Oct4, Klf4, Sox2, c-Myc). These cells has the all characteristic features of human embryonic stem cells (hES) as
self-renewal and ability to differentiate to cells of all three
germ layers. Growing body of published data clearly shows
that that epigenetic mechanisms are involved in the process
of reprogramming of somatic cells into pluripotent state.
TRIM28/KAP1 protein is one of the epigenetic regulators
that regulate heterochromatin formation through histone
modifications and DNA methylation. Over 500 unique zinc
finger proteins containing KRAB domain recruit TRIM28/
KAP1 protein to genomic DNA in sequence-specific fashion thus leading to transcriptional repression of neighboring genes. Here, we showed that TRIM28/KAP1 protein
controls self-renewal of hES and hiPS cells. Stable knockdown of TRIM28/KAP1 gene expression in both hES
and hiPS cells achieved using lentiviral vectors carrying
specific shRNAs resulted in progressive loss of pluripotent
phenotype as confirmed by analysis of surface embryonic
markers expression using immunofluorescence and FACS
analyses. Moreover, we have documented progressive loss
of expression of selected embryonic transcription factors
in hES and hiPS cells depleted of TRIM28/KAP1 expression. Our ongoing experiments are focused on detailed
analysis of the molecular mechanisms that are involved in
the observed phenotype and may shed new light on epigenetic mechanisms that preserve self-renewal of hES and
hiPS cells.
Krzysztof Szade1,2,*, Karolina Bukowska-Strakova1,2,*, Witold
Norbert Nowak,1,2, Agata Szade1,2, Neli KachamakovaTrojanowska1,2, Monika Zukowska1, Sebastian Borys3,
Maciej Małecki3, Alicja Jozkowicz1,#, and Jozef Dulak1,#
1Department of Medical Biotechnology, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland;
2Jagiellonian Centre for Experimental Therapeutics, Jagiellonian
University, Kraków, Poland; 3Department of Metabolic Diseases,
Jagiellonian University Medical College, Kraków, Poland
e-mail: Karolina Bukowska-Strakova <[email protected]>
*These authors equally contributed to the study
#These authors equally contributed as senior authors
It was proposed that adult tissues harbor population of
small pluripotent stem cells. Those cells were reported
to express embryonic marker Oct-4 and are reportedly
described as Very Small Embryonic-Like Cells (VSELs).
However, Oct-4 gene possesses also isoforms and pseudogens that are not markers of pluripotency. In the present study, we examined the expression of Oct-4 isoforms in mouse and human tissues and analyzed stemness
potential of VSEL population.
First, the expression of Oct4-A and Oct-4B mRNA was
investigated in murine bone marrow (BM). There was
no expression of either isoforms in whole bone marrow
as well as in sorted Lin-Sca-1+CD45-FSClow VSELs. Additionally, qRT-PCR done on single sorted cells did not
show presence of Oct-4 expressing cells among Lin-Sca1+CD45-FSClow population.
Next, we verified if VSELs could be detected in human
peripheral blood similarly to other described stem cell
populations. Flow cytometry analysis revealed higher numbers of Lin-CD45-CD34+ VSELs in patients
with type 2 diabetes than in healthy controls. Moreover, number of VSELs correlated with the Hb1Ac and
inversely correlated with endothelial progenitor cells
(CD45dimCD31+CD34+KDR+) and mesenchymal cells
(CD45-CD29+CD90+).
Next, we designed primers allowing discrimination of human Oct-4A, Oct4-B and Oct-4B1 isoforms. The qRTPCR revealed no specific signal for all of these isoforms
in human adult peripheral blood as well as in whole bone
marrow.
Furthermore, we investigated the potential of murine
Lin-Sca-1+CD45- BM-derived VSELs to differentiate
toward hematopoietic cells. Despite the expression of
some markers of hematopoietic stem cells (c-Kit and
CD105) VSELs did not give rise to hematopoietic colonies in single cell colony assays. Moreover, co-culture
with stromal OP-9 cell did not induce the hematopoietic
potential of VSELs.
Next experiments revealed that population of murine
BM-derived Lin-Sca-1+CD45-FSClow is enriched in early
apoptotic cells as shown by high nuclear dye binding and
TUNEL assay. Moreover, ex-vivo enucleation of erythroblasts demonstrated that nuclei expelled from erythroblasts could possess Lin-Sca-1+CD45-FSClow phenotype
and could affect analysis of viable cells among Lin-Sca1+CD45-FSClow population.
To sum up, we did not confirm presence of Oct-4 expressing VSELs in adult humans and mice. Murine BMderived VSELs did not show hematopoietic potential
and are enriched in early apoptotic cells. Our and others
78
analysis [1] suggested lack of stemness potential of human VSELs.
P5.3
Acknowledgements:
Supported by the Innovative Economy Operational Programme Grants
POIG 01.01.02-00-109/09 and 01.01.02-00-069/09.
Increased expression of CD47 and CD274
immunosuppressive proteins on HSCs
after allogeneic HSCs transplantation
Reference:
1. Danova-Alt et al (2012) PLoS One 7.
Agnieszka Ciomber, Iwona Mitrus, Wojciech
Fidyk, Andrzej Smagur, Sebastian Giebel
Maria Skłodowska-Curie Memorial Cancer Center and Institute
of Oncology Gliwice Branch, Department of Bone Marrow
Transplantation, Gliwice, Poland
e-mail: Agnieszka Ciomber <[email protected]>
Hematopoetic stem cells (HSCs) are responsible for the
production of all the lineages of blood cells. HSCs reside
inside the bone marrow’s microevironment, where several
groups of cells and extracellular matrix elements interact
with HSCs to regulate their self renewal, differentiation
and migration. HSCs may directly interact with the immune system. They are capable of protecting themselves
from the attack of both innate and adaptive immune system cells. They possess the ability to regulate expression of
CD47 and CD274 proteins, which are believed to have immunosupressive function. Cell-surface CD47 interacts with
SIRPα receptor on macrophages to inhibit phagocytosis of
HSCs. CD274 inhibits T- cell immunity by induction of
apoptosis in T lymphocytes. Expression of these proteins
on HSCs can be induced by stress or immune signals but
this process is still poorly described.
The aim of the study was to compare the expression of
immunosuppressive proteins CD47 and CD274 on the
surface of HSCs in bone marrow. Samples were obtained
from patients before allogeneic HSCs transplantation and
28 days after the procedure. The analysis were performed
using flow cytometry and appropriate antibodies.
Our results demonstrate increased expression of both
CD47 and CD274 proteins on HSCs in the bone marrow
after HSCs transplantation. The average percentage of
HSCs that express the CD47 protein increases by about
5 percent compared to the values preceding the transplantation. In turn, the percent of CD274 positive HSCs is
2- fold higher than the one before transplantation. These
results indicate a clear change in phenotype of these cells
after HSCs transplantation. We assume that the increased
expression of immunosupresive surface proteins on HSCs
is associated with stress conditions in bone marrow microenvironment after HSCs transplantation. Donor HSCs are
taken out of their natural microenvironment and are exposed to the hostility of recipient’s immune system. The
higher expression of immunosuppressive surface proteins
probably allows survival in recipient’s microenvironment.
CD47 and CD274 surface proteins allow HSCs to adjust to
different environmental conditions. Acknowledgements:
The study was supported by Grant No. 2011/03/B/NZ6/04917 KTS.
P5.4
KRAB-dependent DNA methylation
during reprogramming of human
induced pluripotent stem cells
The histone modifications of the integrated expression
cassette will be further analysed by chromatin immunoprecipitation.
Marta Gładych1, Urszula Oleksiewicz1, Maciej Wiznerowicz1,2
Poznań, Poland; 2Gene Therapy Laboratory, DepartmentCancer
e-mail: Marta Gładych <[email protected]>
Induced pluripotent stem cells (iPSCs) are generated by
reprogramming of somatic cells through the exogenous
expression of specific transcription factors. During reprogramming cells undergo ordered epigenetic alterations including histone modifications and DNA methylation. Our
research interest focuses on a family of zinc finger proteins carrying KRAB domain (KRAB-ZFPs). Binding of
KRAB-ZFPs to DNA in the vicinity of gene promoters
triggers transcriptional silencing through histone modifications that in certain settings leads to DNA methylation.
Here, we aimed to probe if KRAB domain may trigger
DNA methylation of exogenous PGK (phosphoglycerate kinase) promoter during the reprogramming of human primary fibroblasts to iPS cells. We took advantage
of inducible system that relies on conditional, doxycycline
(Dox)-dependent binding of chimeric tTRKRAB transrepressor to tetO element from E. coli tetracycline operator.
In order to engineer reporter cell lines, primary fibroblasts
were transduced with lentiviral vectors carrying PGK-GFP
expression cassette flanked by tetO sequences and cDNA
encoding tTRKRAB. Engineered cells were induced to
pluripotency by forced expression of Oct4, Klf4, Sox2 and
Myc genes and cultured in the presence or absence of doxycycline. Individual iPS colonies were picked up manually
and propagated for further analysis.
First, we performed immunocytochemistry and confirmed
that our iPS colonies express pluripotency markers (NANOG, SSEA4, TRA1-60 and TRA 1-81). The presence of
exogenous PGK and tTRKRAB was confirmed by PCR.
Next, we utilized flow cytometry to measure the expression level of GFP reporter protein and bisulfite sequencing to analyse methylation level of exogenous PGK promoter. Control cells lines that harboured PGK-GFP vector
without tetO sequence exhibited high percentage of GFP
positive cells (97.2–98.6%) and low level of PGK promoter
methylation (1.4–2.8%). In contrast, the cells transduced
with the vector containing PGK-GFP-tetO showed low
expression of GFP protein (0,7%) and hypermethylation
within PGK promoter (98.6%). We added doxycycline to
the cells that were previously cultured without the antibiotic and analysed them after 10 days. No change in GFP
expression and PGK promoter methylation was observed
following incubation with doxycycline. In contrast, in somatic cells addition of doxycyclin restored GFP fluorescence. Our results indicate that during reprogramming
tTRKRAB protein binding to tetO sequence leads to stable
suppression of the transgene, which is not the case in somatic cells.
Artificial system constructed in our laboratory is an efficient tool to study kinetics of DNA methylation during
reprogramming and propagation of iPS cells. Our results
showed that tTRKRAB protein binding to tetO sequence
leads to suppression of transgene. In somatic cells after
doxycycline administration GFP fluorescence is restored
in contrast to iPSCs where it remains low. By administration of doxycyclin we examined that the suppression of
transgene during reprogramming is KRAB-dependent.
80
P5.5
Very small embryonic-like (VSEL)
stem cells — isolation, features and
regenerative potential — an update
Elżbieta Kamycka1, Sylwia Bobis-Wozowicz1, Anna
Łabędź-Masłowska1, Mariusz Z. Ratajczak2, Dariusz
Boruczkowski3, Zbigniew Madeja1, Ewa K. Zuba-Surma1
1Department of Cell Biology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Kraków, Poland; 2Department
of Physiology, Pomeranian Medical University, Szczecin, Poland; 3Polish
Stem Cell Bank, Warsaw, Poland e-mail: Ewa K. Zuba-Surma<[email protected]>
Very small embryonic-like stem cells (VSELs) were identified as rare quiescent population of non-hematopoietic
stem cells (SCs) in adult murine, human and recently rat
tissues including bone marrow (BM) [1-3]. Murine VSELs
were described as small FSClow/CD45-/Lin-/Sca-1+ cells
expressing Oct-4A marker of pluripotency and sharing
several features with embryonic stem cells also on genetic level [4]. Although, due to their unique epigenetic status [4], they are non-proliferative in vitro, VSELs may be
activated in vivo and participate in tissue regenerations as
shown in murine models of tissue injury including heart
[5-6], lungs [7], pancreas [8] and bone [9] as well intermittent hypoxia (IH) [10]. Because of their primitive features
they may also enforce some tumor development incorporating into stroma [11]. Human umbilical cord blood (CB)
is a source of not only hematopoietic stem cells (HSCs)
but also non-hematopoietic SCs including VSELs. CB-derived population of VSELs was characterized as very small
(<7µm), rare, small FSClow/CD45-/Lin-/CD133+ fraction,
possessing large nuclei that contain primitive euchromatin.
Molecular analyses reveal that human VSELs also express
several pluripotent transcription factors like Oct-4A, Nanog, indicating their embryonic-like characteristics [12-13].
Importantly, identification of these cells was possible due
to optimized sorting protocols (FACS method) focused
exclusively on very small FSClow/SSClow objects in gating
strategies [3, 13-14].
In our recent studies, we examined several antigen expression on human and murine VSELs. By using multiparameter flow cytometry (LSR II; BD Bioscience), we quantitatively examined co-expression of CD133 with primary
CD34 and CXCR4 antigens as well as dozens other markers
(>200 by using human Lyoplate system; BD Bioscience) on
CB-derived VSELs. We found that these cells are heterogeneous fraction of non-hematopoietic stem cells, which
can be divided into few subpopulations with distinct coexpression pattern of these crucial three markers. We elucidated that more than half (54 %) of FSClow/CD45-/Lin-/
CD133+ cells may co-express CD34, however only very
small fraction (0,025 %) can co-express CXCR4 antigen.
These flow cytometry data were confirmed using imaging
cytometry (ImageStream system; Amnis Corp.). Thus, human VSELs may not be identified based on CXCR4 expression. Importantly, we confirmed that purified human
FSClow/CD45-/Lin-/CD133+ VSELs are diploid cells with
normal kariotype as confirmed by cell cycle and cytogenetic microarray analysis (CytoScan 750K Array; Affymetrix).
We have also identified potential novel markers identifying
subpopulations of these cells in human specimens. Similarly, we performed extensive evaluation of murine VSELs
heterogeneity screening more than 150 antigens expression on FSClow/CD45-/Lin-/Sca-1+ BM- derived VSELs
(murine Lyoplate platform; BD Biosciences). We selected
few novel markers that may be employed for greater VSEL
purification in the future. Importantly, we found that these
cells may proliferate in vivo in murine model of limb ischemia that is accompanied with visible change in expression of vast markers related to pluripotency (including
Oct-4 and Nanog) and proliferation status of these cells
(e.g. H19 and Igf2).
In conclusion, we have recently extensively evaluated heterogeneity of both murine and human VSELs indicating
few new markers for their better purification. We found
that normal (with normal kariotype), Oct-4A expressing
human VSELs may not be identified by CXCR4 antigen,
but should be isolated as targeted FSClow/CD45-/Lin-/
CD133+ cells via FACS. Similarly, the isolation procedures
of murine FSClow/CD45-/Lin-/Sca-1+ BM- derived VSELs need to be performed based on well validated protocols targeting exclusively small objects. Importantly, we
confirmed that VSELs may be activated in vivo and participate in tissue regeneration. However, the expansion strategies for these cells focusing on epigenetic status modification need to be further optimized to enforce the practical
applications of VSELs in tissue repair.
References:
1. Kucia et al (2006) A population of very small embryonic-like (VSEL)
CXCR4(+)SSEA-1(+)Oct-4+ stem cells identified in adult bone marrow.
Leukemia 20: 857–869.
2. Zuba-Surma et al (2008) Very small embryonic-like stem cells are present in adult murine organs: ImageStream-based morphological analysis
and distribution studies. Cytometry A; 73A: 1116–1127.
3. Zuba-Surma et al (2008) Morphological characterization of Very Small
Embryonic-Like stem cells (VSELs) by ImageStream system analysis. J
Cell Mol Med 12: 292–303.
4. Shin et al (2009) Novel epigenetic mechanisms that control pluripotency and quiescence of adult bone marrow-derived Oct4(+) very small
embryonic-like stem cells. Leukemia 23: 2042–2051.
5. Dawn et al (2008) Transplantation of bone marrow-derived very small
embryonic-like stem cells (VSELs) attenuates left ventricular dysfunction
and remodeling after myocardial infarction. Stem Cells 26: 1646–1655.
6. Zuba-Surma et al (2010) Transplantation of expanded bone marrowderived very small embryonic-like stem cells (VSEL-SCs) improves left
ventricular function and remodeling after myocardial infarction. J Cell Mol
Med Jul 12.
7. Kassmer et al (2013) Very Small Embryonic-Like Stem Cells from the
Murine Bone Marrow Differentiate into Epithelial Cells of the Lung. Stem
Cells May 16.
8. Huang et al (2010) Bone marrow transplantation temporarily improves
pancreatic function in streptozotocin-induced diabetes: potential involvement of very small embryonic-like cells. Transplantation 89: 677–685.
9. Havens et al (2013) Human very small embryonic-like cells generate
skeletal structures, in vivo. Stem Cells Dev 22: 622–630.
10. Gharib et al (2010) Intermittent hypoxia mobilizes bone marrow-derived very small embryonic-like stem cells and activates developmental
transcriptional programs in mice. Sleep 33: 1439–1446.
11. Jung et al (2013) Recruitment of mesenchymal stem cells into prostate
tumours promotes metastasis. Nat Commun 4: 1795.
12. Kucia et al (2007) Morphological and molecular characterization of
novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryoniclike cells purified from human cord blood: preliminary report. Leukemia
21: 297–303.
13. Zuba-Surma et al (2010) Optimization of isolation and further characterization of umbilical cord blood-derived very small embryonic/ epiblast-like stem cells (VSELs). Eur J Haematol 18: 34–46.
14. Zuba-Surma et al (2010) Overview of very small embryonic-like stem
cells (VSELs) and methodology of their identification and isolation by
flow cytometric methods. Curr Protoc Cytom 9: Unit 9.29.
P5.6
P5.7
Role of TRIM28/KAP1 protein in
reprogramming of mouse fibroblasts to
induced pluripotent stem cells (miPSCs)
Acellular proangiogenic therapy
Marta Klimczak1, Anna Misiewicz1, Andrzej
Mackiewicz1,2, Maciej Wiznerowicz1,2
1Gene Therapy
Laboratory, Department of Cancer Immunology,
e-mail: Marta Klimczak <[email protected]>
Mouse induced pluripotent stem cells (miPSCs) can be generated from mouse somatic cells by forced over-expression
of embryonic transcription factors that most commonly
involve: Oct4, Sox2, Klf4 and c-Myc. miPSCs are highly
similar to embryonic stem cells (ESCs) with reference to
proliferation and differentiation capacity. Therefore, they
provide a promising source of pluripotent cells for not
only basic stem cell biology but also clinical cell-based therapies and disease modeling. The major goal of this study is
to determine the role of KAP1 protein in reprogramming
and self-renewal of miPSCs. TRIM28/Kap1 is a cofactor
of KRAB-containing zinc finger proteins (KRAB-ZFPs).
These are known as the transcriptional repressors which
act by formation of heterochromatin through histone
modifications, HP1 binding and DNA methylation. In the
first step, TRIM28/Kap1 expression in primary mouse embryonic fibroblasts (MEF) was silenced by a mean of lentiviral vectors carrying short hairpin RNA (shRNA) specific
for TRIM28/Kap1. Next, the cells were reprogrammed to
miPSC using lentiviral vector carrying Oct4, Sox2, Klf4 and
c-Myc transgenes. Immunofluorescence analysis of embryonic markers revealed that TRIM28/Kap1 knockdown results in accelerated reprogramming of mouse fibroblasts to
iPS cells. Our preliminary results suggests that repressive
chromatin state imposed by TRIM28/Kap1 prohibits the
reprogramming thus helps to maintain identity of differentiated cells. M. Kozakowska1,2, J. Kotlinowski1, M. Ciesla1, A.
Stefanska1, B. Pilecki1, A. Grochot-Przeczek1,2,
R. Derlacz3, J. Dulak1, A. Jozkowicz1
1Department of Medical Biotechnology, Faculty of biochemistry,
Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland;
2Jagiellonian Centre for Experimental Therapeutics, Jagiellonian
University, Kraków, Poland; 3R&D Department, ADAMED Ltd, Pienków,
Poland
e-mail: Magdalena Kozakowska <[email protected]>
Heme oxygenase 1 (HO-1), a heme-degrading enzyme,
was recently found to increase viability and proliferation
of skeletal muscle progenitor cells (satellite cells and myoblasts of C2C12 cell line) (Jazwa et al., 2013; Kozakowska
et al., 2012). Concomitantly and unexpectedly, we have also
shown, that HO-1 overexpression reduced potently differentiation of C2C12 cells, while primary muscle satellite cells isolated from mice lacking HO-1 showed accelerated differentiation (Kozakowska et al., 2012). HO-1 is
also proangiogenic (Dulak et al., 2008; Jazwa et al., 2013),
and accordingly, disturbed wound healing in diabetes may
be caused by its decreased expression in the skin (GrochotPrzeczek et al., 2009). Therefore, the aim of this study was
to examine the paracrine effect of HO-1overexpressing
myoblast on blood flow restoration after hind-limb ischemia in diabetic mice.
Retroviral overexpression of HO-1 in C2C12 cells led
to increased production of proangiogenic SDF-1a, and
myoblasts differentiation potentiated this effect. In comparison to control cells, HO-1 diminished ROS production and mortality after H2O2 treatment. Upon injection
into NOD/SCID mice C2C12 cells overexpressing HO-1
did not differentiate and formed sarcomas. In contrast,
control cells were able to convert into mature myotubes.
Interestingly, administration of conditioned media from
myoblasts expressing HO-1 into ischemic muscle of diabetic dbdb mice improved the blood flow restoration on
28th day after surgery (measured by Laser Doppler system),
without inducing inflammation and neoplastic growth. Immunofluorescent visualization of CD31 in muscle cryosections revealed increased vessel number in mice treated
with conditioned media from myoblasts expressing HO-1.
Real-time RT-PCR showed increased gradient of SDF1 between muscle and bone marrow. In spite of that, the
number of putative endothelial progenitor cells (Sca-1+,
CD45_, VEGFR2+) was changed between groups neither
in bone marrow nor in peripheral blood
The acellular strategy, involving conditioned media from
cells overexpressing HO-1 improves muscle blood flow
restoration after ischemia in dbdb mice. Such an approach
could be safer than the cell therapy treatment of non-healing diabetic wounds.
Acknowledgements:
This work was supported by grants POIG 01.01.02-00-109/09 and
01.01.02.069/09 from the European Union structural funds.
References:
Dulak J et al (2008) Heme oxygenase-1 and carbon monoxide in vascular
pathobiology: focus on angiogenesis. Circulation 117: 231–241.
Grochot-Przeczek A et al (2009) Heme oxygenase-1 accelerates cutaneous
wound healing in mice. PLoS One 4: e5803.
Jazwa A et al (2013) Pre-emptive hypoxia-regulated HO-1 gene therapy
improves post-ischaemic limb perfusion and tissue regeneration in mice.
Cardiovasc Res 97: 115–124.
Kozakowska M et al (2012) Heme oxygenase-1 inhibits myoblast differentiation by targeting myomirs. Antioxid Redox Signal 16: 113–127
82
P5.8
P5.9
Role of epigenetic regulator-KAP1/
TRIM28 in reprogramming of human
induced pluripotent stem cells (iPS)
Comparison of methods for
proliferative activity assessment in
chondrocyte spatial cell cultures
Katarzyna Kulcenty1,2, Urszula Oleksiewicz1,2,
MAciej Wiznerowicz1,2
Anna Kulczycka, Arkadiusz Orchel, Zofia Dzierżewicz
1University
of Medical Sciences, Chair of Medical Biotechnology,
Department of Cancer Immunology, Poznań, Poland; 2Greater Poland
Cancer Centre, Department of Cancer Immunology, Poznań, Poland
e-mail: Katarzyna Kulcenty <[email protected]>
Induced pluripotent stem cells (iPS) are derived from somatic cells through ectopic expression of few embryonic transcription factors. They have two unique properties:
self-renewal and pluripotency, i.e. ability to differentiate to
almost every cell of the adult organism. The broad potential of the iPS cells technology can be applied in regenerative medicine and/or basic research to study fundamental
mechanisms of mammalian development. Results of the
recent studies of human iPS cells revealed that epigenetic
mechanisms are involved in the process of reprogramming
of somatic cells into pluripotent state. KAP1 protein is one
of the epigenetic regulators that regulate heterochromatin
formation through histone modifications and DNA methylation. Here, we probed a potential role for KAP1 protein
during reprogramming of human primary fibroblasts to
iPS cells. In the first series of experiments, we achieved efficient and stable knockdown of KAP1 gene expression in
human primary fibroblasts using lentiviral vectors carrying
specific shRNAs. Next, the modified fibroblasts were transduced with lentiviral vector carrying Oct4, Sox2, Klf4 and
c-Myc transgenes and reprogrammed to iPS cells. Pluripotent phenotype of the obtained iPS colonies was confirmed
by analysis of surface embryonic markers expression using
immunofluorescence and FACS. The number of emerging
iPS colonies was significantly higher when KAP1 gene expression was specifically silenced during the early stages of
reprogramming process. Importantly, the colonies of pluripotent cells appeared much faster in the absence of KAP1
expression. Our preliminary results strongly suggest that
knockdown of KAP1 gene in human primary fibroblasts
facilitates formation of human iPS cells, suggesting a novel
role for KAP1 during reprogramming of human somatic
cells. Our ongoing experiments are focused on detailed
analysis of the molecular mechanisms that are involved in
the observed phenotype and may shed new light on dynamic of epigenetic modifications during cellular reprogramming.
Department of Biopharmacy, School of Pharmacy with Division of
Medical Analytics, Medical University of Silesia in Katowice, Katowice,
Poland
e-mail: Anna Kulczycka <[email protected]>
Cartilage has a limited ability for self-repair. This limitation
drives a development of cartilage tissue engineering. Tissue engineering uses cells placed in tridimensional scaffolds
to enable appropriate growth of cells and mimic natural
tissue conditions. The drawback of scaffold using is its
heterogeneity that may cause an increased risk of over- or
underestimation of cell proliferative status. To solve this
problem it is necessary to use markers that assess accurate
and reliable status of cells, based on eg. expression levels
of genes coding proteins involved in DNA replication like
histone H3/f.
The aim of the study was to compare results obtained using commercial cell proliferation assays (XTT and resazurin
based) with expression levels of histone H3/f. The XTT
and resazurin dyes were used to compare a method based
on molecular biology (histone H3/f expression) with the
measurements of cell metabolic activity. The resazurin
assay estimates a bioreduction of the dye in proliferating
cells, manifested by a decrease in the absorbance at 490 nm.
XTT reduction by mitochondrial dehydrogenase results in
an increase in the absorbance at 450 nm. The accuracy of
both methods is limited by the test compound diffusion
rate into the scaffolds and the degree of scaffold's heterogeneity.
The experiment was performed on human articular chondrocytes. Cells were growing on a standard plastic substrate
(control 2D culture), in agar and fibrin glue. Control group
included cells growing in typical monolayer conditions.
Histone H3/f expression and cell metabolic activity were
determined in the cells having various proliferative activity:
growing exponentially and growth-arrested. The assessment of histone H3/f expression was done by the use of a
real-time RT-PCR technique.
Acknowledgements:
The work was supported by KNW-1-036/D/2/0.
P5.10
P5.11
Differentiation of induced pluripotent stem
cells (iPSC) into functional cardiomyocytes
TRIM28/KAP1 triggers DNA methylation
of cellular promoters during
reprogramming of human fibroblasts
to induced pluripotent cells
Sylwia Mazurek1, Maciej Wiznerowicz1,2
e-mail: Sylwia Mazurek <[email protected]>
Efficient differentiation of pluripotent stem cells (iPS) to
functional cardiomyocytes (CM) offers various opportunities including: patient-specific cardiac regeneration, disease
modelling and cardiotoxicity drug testing. iPS cells are generated by reprogramming of somatic cells to pluripotency
through forced expression of embryonic transcription factors and serve as potentially unlimited source of various
cell types that can be used in regenerative medicine. In order to induce cardiomyocyte differentiation from human
iPSC, we used method based on forced aggregation embryoid bodies (EBs) formation in medium supplemented with
ascorbic acid and bone morphogenetic protein 4 (BMP4).
First contracting areas appeared after 10 days of differentiation. Obtained cardiomyocytes formed contracting myotubes after 35 days. Immunocytochemical staining confirmed presence of cardiac markers (Troponin T, GATA4).
The CM differentiation was confirmed by expression
analysis of selected markers for: embryonic cells (OCT4,
SOX2, NANOG), mesoderm (T, MESP1) and cardiomyocytes (TNNT2, MYH6). The results of our studies can
be applied for generation of functional human CM from
patient-specific iPS cells and pave the way for regeneration
of heart muscle injury in future.
Marta Gładych1, Urszula Oleksiewicz1, Maciej Wiznerowicz 1,2
Poznań, Poland; 2Gene Therapy Laboratory, DepartmentCancer
e-mail: Urszula Oleksiewicz <[email protected]>
Induced pluripotent stem cells (iPSCs) are generated by
dedifferentiation of adult cells through forced expression
of few embryonic transcription factors. Reprogramming
involves ordered and global epigenetic alterations including
histone modifications and DNA methylation that progressively silence expression of lineage-specific genes whilst
enabling transcription of embryonic factors. TRIM28/
KAP1 protein is a potent epigenetic modulator that induces heterochromatin formation when recruited to DNA in
a sequence-specific fashion by zinc finger proteins containing KRAB domain, thus, leading to transcriptional repression of neighbouring genes through histone modifications
and HP1 recruitment. Exclusively during early embryonic
development, TRIM28/KAP1-mediated epigenetic repression leads to DNA methylation that is largely restricted to
repetitive elements thus protecting genome stability. Here,
we have shown that TRIM28/KAP1 triggers DNA methylation of cellular promoters during reprogramming of
human primary fibroblasts to iPS cells. Conditional repression of PGK promoter was achieved by chimeric transrepressor (tetRKRAB) containing KRAB domain fused with
DNA binding domain of tetracycline repressor (tetR) that
binds to tetO element from E. coli tetracycline operator
in doxycycline (dox)-controllable manner. The tetRKRAB
transgene and tetO-PGK-GFP expression cassette were
stably integrated into genome of human primary fibroblasts using lentiviral vectors. Next, the engineered cells
were reprogrammed to pluripotency by forced expression
of Oct4, Klf4, Sox2 and Myc genes, either in the presence
(dox+) or absence (dox-) of doxycycline. Phenotype of obtained iPS cell lines was confirmed by immunoflurescence
analysis of expression of selected embryonic markers (NANOG, SSEA4, TRA1-60 and TRA 1-81). Bisulfite sequencing of PGK promoter that was subjected to tetKRAB-mediated epigenetic repression during reprogramming in the
absence of dox revealed high level of CpG methylation
(98.6%). In contrast, PGK promoter in human iPS cells
obtained from the engineered fibroblasts in the presence
of dox that sequestrated tetRKRAB from the tetO-PGKGFP cassette remained largely unmethylated (1.4–2.8%).
Our results strongly suggest that transcriptional repressors
containing KRAB domain may be involved in changing
landscape of genomic DNA methylation during cellular
reprogramming.
84
P5.12
P5.13
New isolation approach of murine bone
marrow-derived mesenchymal stem
cells retrieving fraction with wider
proliferation and differentation capacity
Reactive oxygen species stimulate
TGFbeta expression during
osteogenic differentiation
Agnieszka Pająk, Anna Łabądz-Masłowska,
Zbigniew Madeja, Ewa Zuba-Surma
Department of Cell Biology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Kraków, Poland
e-mail: Agnieszka Pająk <[email protected]>
Agnieszka Robaszkiewicz1,2, László Virág1,3
1Department of Medical Chemistry, Medical and Health Science
Center, University of Debrecen, Debrecen, Hungary; 2Department
of Environmental Pollution Biophysics, Faculty of Biology and
Environmental Protection, University of Lodz, Łódź, Poland; 3MTA DE
Cell Biology and Signaling Research Group, Debrecen, Hungary
e-mail: Agnieszka Robaszkiewicz <[email protected]>
Bone marrow-derived mesenchymal stem cells (BMMSCs)
represent stem cell population interesting for regenerative
medicine due to their multilineage differentiation capacity,
mediated paracrine effect and low immunogenicity. Previously established protocols for isolation of these cells rely
on flushing out of bone marrow tissue from bone cavity.
In this work, we modified the isolation method by employing additional enzymatic steps. According to the new approach, previously rinsed and chopped femora and tibias
from four-week-old mice, were additionally treated with
collagenases I+II solution. Efficacy of both extraction
methods (“flushed+digested”; FD and “flushed only”;
Fo) and phenotype of isolated fractions of BMMSCs were
monitored by microscopy, flow cytometry phenotyping and
gene expression evaluation by quantitative real time PCR.
We found that cells obtained only by flushing bones have
lower proliferation rate in vitro and stopped growth after
about 5 passages, in contrary to cells obtained by flushing
plus enzymatic digestion, which reached more than 10 passages and divided more intensively during the entire culture time. Significant morphological differences were also
observed between cells isolated by FD and Fo method.
Moreover, gene expression analysis showed up-regulaton
of pluripotency genes as Nanog or Oct-4 in FD fraction,
what wasn’t seen for Fo - derived cells. Flow cytometry
phenotyping was also performed showing 1) gradual loss
of CD45 experssion over culture for FD cells; 2) enrichment in vast MSC markers (CD90,CD105) for FD cells,
and 3) lack of endothelial (CD144, Flk-1) and epithelial
(CD324) markers, in both fractions.
Summarizing, new isolation approach of BMMSCs provides greater yield and releases of their distinct fractions
from periosteum niches. Such cells drive cell growth of
culture in vitro, provides higher proliferation rate and might
have wider differentiation capability which needs to be further investigated.
Transforming growth factor beta belongs to the family of
cytokines affecting various cellular processes such as proliferation, differentiation and death in an autocrine as well
as paracrine fashion. Using well-established cellular models
of SAOS2 (osteosarcoma cell line) and mesenchymal stem
cells (MSCs) isolated from human placenta we decided
to verify hypothesis about putative involvement of reactive oxygen species in the intracellular signaling cascades
leading to expression of TGFbeta during osteogenic differentiation. We found that expression of NADPH oxidase isoform 4 (NOX4) strongly correlated with gradually
increasing total NOX activity, while the level of NOX2
mRNA was only slightly elevated and NOX1 mRNA was
not detectable. These data suggest that NOX4 may be the
main source of differentiation-associated ROS generation.
Augmented ROS release was accompanied by the increase
in TGFbeta expression, activation of the redox sensitive
MAP kinase p38 and transcriptional activation of AP-1.
Supplementation of osteogenic medium with catalase or
the p38 inhibitor SB203580 prevented phosphorylation of
p38 and significantly reduced the level of TGFbeta mRNA
suggesting that p38 is the missing link between ROS production and TGFbeta expression.
References:
Beyer Nardi N, da Silva Meirelles L (2006) Mesenchymal stem cells:
Isolation, in vitro expansion and characterization. Handb Exp Pharmacol
249–282.
P5.14
P5.15
Transcriptomic signature of tissue
injury: Meta-analysis of microarray
gene expression data
Nucleotide metabolism in human
adipose derived stem cells (hADSCs)
Piotr Sass, Justyna Podolak, Bartosz
Górnikiewicz, Paweł Sachadyn
Gdańsk University of Technology, Microbiology Department, Gdańsk,
Poland
e-mail: Piotr Sass <[email protected]>
The complexities of the wound healing and regeneration
processes make them difficult to understand and to control.
The introduction of gene expression profiling by array has
enabled the observation of global expression changes. Vast
amount of microarray data on gene expression following
injury have been collected so far. This study is an attempt
to identify the genes which exhibit remarkable changes in
gene expression in response to injury in different tissues.
To our knowledge this is the first meta-analysis of gene
expression profiles in wound area. The available microarray
data on gene expression in response to tissue injury were
downloaded from Gene Expression Omnibus Database
(GEO). The analysis includes 39 microarray experiments
reported in several independent studies and performed for
as different tissues as heart, liver, skin, bones, and spinal
cord, collected from rat, mouse and human. The genes displaying at least two-fold increase in expression in response
to injury in at least half of microarray experiments in the
analysis as well as those displaying at least two-fold decrease in expression in at least a third of the microarray experiments were singled out in order to reveal the lists of 13
up-regulated and 17 down-regulated genes. This selection
of genes may be considered as a transcriptomic signature
of tissue injury.
Interestingly, the prevailing number of the down-regulated
genes is not repressed in response to heart injury in the
MRL/MpJ mouse, an inbred strain exhibiting enhanced
regeneration potential. The quantitative Real-Time PCR
examination confirmed that the transcript level of the Pah
gene encoding phenylalanine hydroxylase, which belongs to
the group of 17 genes repressed following tissue injury, is
two orders of magnitude higher in the heart of the MRL/
MpJ than that in that of the normal C57BL/6J mouse.
The gene set enrichment analysis performed using Molecular Signature Database (Broad Institute, Boston) showed
that seven out of the group of seventeen repressed genes
are regulated by c-Jun N-terminal kinase 1 (JNK1). The
results of the study indicate that response to wounding in
different tissues, species and types of injury may be dependent on a common regulatory mechanism.
Alicja Sielicka1, Ewa Kaniewska1,2, Maciej Śledziński3,4,
Janusz Zdzitowiecki5, Iwona Pelikant-Małecka1,
Magdalena Górska6, Magdalena Zabielska1, Mariola
Olkowicz1, Ewa M. Słomińska1, Ryszard T. Smoleński1
1Medical University of Gdansk, Department of Biochemistry, Gdańsk,
Poland; 2Heart Science Centre, Imperial College London - Harefield
Hospital, United Kingdom, 3Medical University of Gdansk, Department
of General, Endocrine and Transplant Surgery, Gdańsk, Poland; 4Medical
University of Gdansk, Department of Emergency Medicine, Gdańsk,
Poland; 5Sopocka Fabryka Urody, Sopot, Poland; 6Medical University of
Gdansk, Department of Medical Chemistry, Gdańsk, Poland
e-mail: Alicja Sielicka <[email protected]>
Introduction: Intra- and extracellular enzymes of nucleotide metabolism are important not only in the energy metabolism but also in the regulation of the immune response,
inflammation and thrombosis. The aim of our research was
to analyze the level of intra- and extracellular enzymes and
nucleotides in human adipose derived stem cells (hADSCs)
We have investigated level of nucleotides: ATP, ADP, NAD
and NADP and activity of extracellularon enzymes: adenosine deaminase (ADA), ecto-5'-nucleotidase (e5’N), ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Also we measured activity of intracellular enzymes:
purine nucleoside phosphorylase (PNP), adenosine deaminase (ADA) and adenosine monophosphate deaminase
(AMPD) in human adipose derived stem cells (hADSCs). Methods: In this study was used human adipose derived
stem cells (hADSCs). Cells were incubated for 24h in
Mesenchymal Stem Cell Growth Medium DXF at 37°C,
5% CO2. After incubation cells and incubation medium
were separated and analysed for concentration ATP, NAD,
NADP and intra- and extracellular enzymes (ADA, e5’N,
E-NTPDase, PNP, AMPD) using liquid chromatography
with UV detection.
Results: Level of: ATP (22.1±0.8 nmol/mg), NAD
(13.1±1.1 nmol/mg), NADP (0.9±0.4 nmol/mg) (n=9);
extracellular enzymes: ADA (5’) 31.1±13.9 µM/min./
mg; e5’N (15’) 43.2±3.2 µM/min/mg; E-NTPDase (15’)
6.7±0.9 µM/min/mg (n=3) and intracellular enzymes:
PNP (15’) 896.1±43.3 µM/min/mg; ADA (30’) 254.5±56.0
µM/min/mg and AMPD (30’) 1819.0±262.1 µM/min/mg
(n=9).
Conclusion:We have demonstrated that human adipose
derived stem cells demonstrate relatively high concentration of NAD and very low level of NADP. Extracellular
enzyme activity of e5'N is higher than in other cell types
and ADA much lower.
86
P5.16
Inducible pluripotent stem cells as a
tool in articular cartilage regeneration
Wiktoria M. Suchorska1, Tomasz Trzeciak2,
Magdalena Richter2, Jarosław Pawlicz2
Poznań University of Medical Sciences. We are going to
examine the mechanism of repair of so-called full thickness cartilage damage which penetrates subchondral bone.
The results of the experiments will form the basis for the
development of regenerative medicine and may contribute
to the development of clinical protocols for future acquisition of chondrocytes from the patient's somatic cells.
1The Greater Poland Cancer Centre, Department of Medical Physics,
Radiobiology Lab, Poznań, Poland 2University of Medical Science,
Department and Clinic of Orthopedics Surgery and Traumatology,
Poznań, Poland
e-mail: Wiktoria M Suchorska <[email protected]>
The main objective of the study was to understand the molecular mechanisms and signal transduction pathways involved in the differentiation process of the stem cells (embryonic human stem cells - hES, and induced pluripotent
stem cells - IPS) into chondrocytes. Specific objectives include:
1. Analysis of the molecular mechanisms involved in differentiation hES cells, and IPS cells into chondrocytes by
analysis of global gene expression profiles at various stages
of cell differentiation
2.Comparison of the mechanisms and signal transduction
pathways involved in differentiation process of hES, and
IPS cells to chondrocytes
3. Multidimensional comparative analysis in vitro of mature
chondrocytes differentiated from both hES, and IPS cells
4. Demonstration the functionality of the hES, and IPS
cells-derived chondrocytes in the regeneration of damaged
articular cartilage in vivo in an animal experimental model
Since 2010, in collaboration with the Laboratory of Gene
Therapy, headed by Dr. M. Wiznerowicz we perform the
research project, which is successfully used a protocol
based on this year's Nobel Prize winning Takahashi and
Yamanaka's experience of introducing into the genome
cells four transcription factors: Oct3/4, Klf4, Sox2 and
cMyc using a viral vector. The results, which we obtained,
confirm the possibility of maintaining in vitro full-featured
IPS cells. In addition, we have established a line of human
embryonic stem cells (hES). Up to the date it was proved
that hES have the capacity to differentiate directly toward
chondrocytes under defined cell culture conditions. During this process we controlled the morphology of the cells,
and the level of gene expression typical for embryonic cells
(OCT4, SOX2, NANOG), for mesoendoderm (MIXL1),
for mesoderm (PDGFRbetha), for endoderm (SOX17,
GATA4) and for mature chondrocytes (SOX9, CD44, collagen type II1). Moreover at the entire process of reprogramming and the differentiation of the cells culture the
population of differentiated cells was examined using immunofluorescence, and immunohistochemistry (safranin O,
collagen type II). We are going to perform a global analysis
of gene expression at different stages of hES, and IPS cells
differentiation. These analyzes based on RNA microarray
technology will be made in
collaboration with the Institute
of Applied Cancer Center, MD Anderson, University of
Texas. The experience gained by the project manager in the
TCGA (Tumor Cancer Genome Atlas) Researches enable
the validation of the most important genes involved in the
molecular mechanisms controlling stem cells differentiation using RT-PCR. In a further step we are going to study the protocol for differentiation IPS cells toward chondrocytes. Obtained chondrocytes will be used as a transplants for repairing cartilage
damage in vivo according to the previously developed methodology. The model of in vitro analysis of regeneration the
articular cartilage has been developed. Animal study are
performed in collaboration with team of Dr Trzeciak from
Department and Clinic of Orthopedics and Traumatology,
P5.17
P5.18
The influence of interleukin-4 on myogenic
potential of pluripotent stem cells Influence of physical exertion on changes
in concentration of sphingosine-1phosphate and the components of the
complement system (C3a, C5a, C5b9) in
healthy volunteers. Preliminary reports
Barbara Świerczek, Jacek Neska, Maria A.
Ciemerych, Karolina Archacka
Department of Cytology, Faculty of Biology, University of Warsaw,
Warsaw Poland
e-mail: Barbara Świerczek <[email protected]>
Pluripotent stem cells such as embryonic stem cells (ES
cells) are characterized by the ability to self-renew and to
differentiate into any given tissue. For this reason they are
considered as a potential universal source of various cell
types for regenerative medicine. In vivo, i.e. in chimeric animals or in teratomas, pluripotent stem cells can differentiate into skeletal muscle tissue. Thus, they are characterized
by myogenic potential, i.e. ability to form functional myogenic cells. However, despite accumulating knowledge no
safe, efficient and reproducible protocol for in vitro generation of myogenic cells from pluripotent stem cells has been
derived so far (Grabowska et al., 2012). Designing of such
method is a crucial task for the development of cell-based
therapy of still incurable skeletal muscle diseases, such as
muscular dystrophies. We have recently showed that undifferentiated mouse ES cells are able to fuse with myoblasts
and participate in myotubes formation. ES cells that fused
with myoblasts downregulate the expression of pluripotency markers and induce the expression of myogenic ones.
However, hybrid myotubes formed by both ES cells and
myoblasts occur with very low frequency.
In this study we investigated whether interleukin-4 (IL-4)
promotes fusion between ES cells and myoblasts. IL-4 was
described as the factor which plays the key role in recruiting myoblasts to myotubes (Horsley et al., 2003). Our preliminary results confirmed that administration of IL-4 in
myoblasts culture results in accelerated formation of myotubes and significantly increases the index of fusion, which
serves as the indicator of cells ability for fusion. We did
not observe any influence of IL-4 on myoblasts proliferation and morphology. We found that both subunits of IL-4
receptors, IL-4Rα and IL-13Rα, were expressed both on
mRNA and protein level in myoblasts, myotubes, and also
in undifferentiated ES cells. This indicated that ES cells
may respond to IL-4 treatment. In the next step we cocultured ES cells and myoblasts in the presence of IL-4 and
found that the number of hybrid myotubes was two times
higher when compared to control co-cultures. However, as
IL-4 treatment also resulted in the increase of index of fusion, the contribution of hybrid myotubes to total number
of myotubes was similar in both types of the co-culture, i.e.
treated with IL-4 and control ones. Marta Budkowska1,2 , Daria Sałata1,2,Ewelina
Żyżniewska-Banaszak3, Barbara Dołęgowska1,2
1Department of Physiology, Laboratory of Physiology and Biochemistry
of Stem Cell, 2Department of Medical Analytics, 3Department of
Physiotherapy and Wellness, Pomeranian Medical University, Szczecin,
Poland
e-mail: Marta Sylwia <[email protected]>
Introduction: Activation of the complement system is
caused by variety of factors: stress, injuries, organs ischemia and certain drugs and chemicals. Various studies
indicated also potential involvement of physical exertion
as an incentive stimulating the activation of the complement system, which interacts with nascent membrane
attack complex (MAC/C5b-9), therefore inducing the
release of sphingosine-1-phosphate (S1P) from erythrocytes. Recent studies have shown that S1P is a main
chemotactic factor, which participates in the process
of stem cells release from bone marrow to peripherial
blood.
Aim of the study: Analysis of concentration of sphingosine-1-phosphate and the complement system components
in healthy volunteers and comparison influence of physical exertion on changes within these factors in volunteers
under and over 50 years of age.
Materials and Method: Blood samples were collected
from 18 healthy volunteers of two age groups: 50 years
old (13 volunteers). Volunteers exercise capacity and exercise tolerance was assessed by exercise test on a treadmill according to the Bruce protocol and in accordance
with current standards. Peripheral blood was collected at
three different times: immediately before exercise test,
three hours after the exercise test, and 6 hours after the
exercise test. Plasma and serum were obtained by centrifuging blood samples (2600 rpm, 10 minutes) and stored
at –20°C until assay. S1P was extracted from plasma by
a one-step method and coupled with o-phtaladialdehyde
(OPA), and the resultant fluorescent derivative was separated by RP-HPLC. S1P-C17 was used as the internal
standard. The quantitation was based on peak areas with
internal standard calibration. The components of the
complement system (C3a, C5a, C5b-9) in serum were
identified by commercially available immunoenzymatic
ELISA test. The results were statistically analyzed using
nonparametric tests (ANOVA, U Mann-Whitney test), at
a given ratio of p<0.05
Results: The only statistically significant differences between the groups (50 years of age) were demonstrated in
concentration of C3a (p=0.004). This was associated with
an increase in the average concentration of C3a in group
50 years of age (p=0.01). In other analysed factors: C5a,
C5b-9, S1P, no statistically significant differences has been
showed between groups during the test.
Conclusion: Physical exertion has a significant impact on
changes in the concentration of anaphylatoxins C3a. No
activation of the other complement system components
indicates that the physical exertion intensity does not cause
erythrocytes hemolysis, and thus S1P, which is stored by
red blood cells, is not released to the peripheral blood.
88
P5.19
P5.20
Dynamics of retroelements activity during
reprogramming of human fibroblast to
induced pluripotent stem cells (iPSc)
Application of epigenetic switch for
generation of human IPS cells
Katarzyna Tomczak1,2, Urszula Oleksiewicz2,
Maciej Wiznerowicz2,3
1Postgraduate School of Molecular Medicine, Medical University of
Warsaw, Warsaw, Poland; 2Laboratory of Gene Therapy, Department
of Cancer Immunology, The Greater Poland Cancer Centre, Poznań,
Poland; 3Department of Cancer Immunology and Diagnostics, Chair of
Medical Biotechnology, Poznan University of Medical Sciences, Poznań,
Poland
e-mail: Katarzyna Tomczak <[email protected]>
Joanna Wróblewska1, Katarzyna Kulcenty1, Urszula
Oleksiewicz1, Wiktoria Suchorska1,2, Jannik Andersen 4, Giulio
Draetta4, Gustavo Mostoslavsky3, Maciej Wiznerowicz1,2
¹Poznan University of Medical Sciences, Chair of Biotechnology, Dept
of Cancer Immunology, Gene Therapy Laboratory, Poznań, Poland;
²Greater Poland Cancer Centre, Dept of Cancer Diagnostics and
Immunology, Gene Therapy Laboratory, Poznań, Poland; ³Boston
University School of Medicine, Dept. of Medicine, Boston, MA, USA;
4Institute for Applied Cancer Science, University of Texas MD Anderson
Cancer Center, Houston, TX, USA
e-mail: Joanna Wróblewska <[email protected]>
Retroelements constitute around 90% of all transposable
elements in the human genome. These endogenous parasite sequences are able to move through the genome from
one location to another contributing to genome diversity
and disease, including cancer. The expression of mobile
elements is regulated by epigenetic mechanisms including
histone modifications and DNA methylation. Intensive
epigenetic remodelling of the chromatin structure is important process occurring during cellular reprogramming
and formation of induced pluripotent stem cells (iPS)
through forced expression of selected transcription factors.
Here, we investigated the dynamics of retroelement activity
during generation of induced pluripotent stem cells (iPS).
Our research including molecular and biochemical analysis
confirmed that cellular reprogramming process induces activation of retroelements (such as LINE-1) in human cells.
In order to obtain insight into epigenetic mechanisms of
the observed phenomena we analysed the role of chosen
protein in regulation of retroelements activity during formation of human iPS cells. Our ongoing experiments are
aimed towards profiling of dynamics of retroelements activity during reprogramming of human cells depleted of
selected epigenetic factors gene expression. Our project
will contribute to better understanding of the molecular
mechanisms that control retroelements during cellular
reprogramming and are involved in host defence against
these genomic parasites. Human induced pluripotent stem cells (IPS) are generated by reprogramming of somatic cells through enforced
expression of embryonic transcription factors. However,
clinical applications require that expression of introduced
transgenes must be permanently switched off in the IPS
cells and obtained differentiated progenies. Here, we took
advantage of epigenetic switch that relies on reversible
binding of tTRKRAB transrepressor to tetO element,
which results in tight transcriptional repression of proximal
promoter through heterochromatin formation. In order to
apply this system for reprogramming, the tetO element
was inserted into pSTEMCCA lentiviral vector carrying
OCT4, SOX2, KLF4 and cMYC under control of EF1alpha promoter. Transduction of human skin fibroblasts
with obtained pSTEMCCA-tetO allowed for expression
of reprogramming factors and thus efficient generation of
human iPS clones. Obtained clones were picked and further cultured until establishing stable IPS cell lines. Then
cells were then transduced with lentiviral vector pLV-HK
carrying tTRKRAB, in order to switch off reprogramming
transgene expression.
Tight repression of introduced transgenes in all human IPS
clones was analyzed by RT-PCR and confirmed full functionality of our system. Obtained IPS cell lines showed no
abnormalities in karyotypes. Pluripotent phenotype of IPS
cells was revealed by analysis of endogenous embryonic
genes expression using RT-PCR and immunofluorescence
staining. Analyzed cells were also able to form embryonic
bodies in vitro and teratomas in immunocompromised
mice, which proved their ability to differentiate into cells
derived from three germ layers. tTRKRAB-mediated epigenetic repression persisted through prolonged culture of
obtained IPS cell lines. Importantly, expression of introduced transgenes remained undetectable after differentiation in vivo.
Our results confirm that our epigenetic switch effectively
prohibits re-expression of embryonic transgenes in human
IPS cells and their differentiated progenies paving the way
for their applications in various fields of regenerative medicine, disease modeling and drug discovery. 48th Congress of the Polish Biochemical Society
P5.21
P5.22
Expression of GFP-myosin VI transgene
induces subtle ultrastructural
changes in Drosophila melanogaster
maturing spermatids that do not
affect individualization process
Ischemic limb injury activates proliferation
and mobilization of bone marrow- derived
very small embryonic-like stem cells
Przemysław Zakrzewski, Marta Lenartowska
Nicolaus Copernicus University, Faculty of Biology and Environmental
Protection, Laboratory of Developmental Biology, Toruń, Poland
e-mail: Przemysław Zakrzewski <[email protected]>
Anna Łabędź-Masłowska1, Elżbieta Kamycka1,
Dominika Berdecka1, Mariusz Z. Ratajczak2,
Zbigniew Madeja1, Ewa K. Zuba-Surma1
1Department of Cell Biology, Faculty of Biochemistry, Biophysics and
Biotechnology, Jagiellonian University, Kraków, Poland; 2Stem Cell
Biology Institute, University of Louisville, Louisville, KY, USA
e-mail: Anna Łabędź-Masłowska <[email protected]>
The green fluorescent protein (GFP), originally cloned
from the jellyfish Aequorea victoria, absorbs blue light and
emits green fluorescence which is stable, species independent, and does not require any substrates or cofactors. GFP retains fluorescence when fused to another
protein, which makes it an attractive fluorescent tag to
monitor subcellular activities such as gene expression,
protein-protein interactions, trafficking and localization
in vivo in real time. Therefore GFP is most widely used as
a live cell reporter in different cell types and organisms,
including mice, zebrafish and Drosophila. It is believed
that GFP-transgene expression is innocuous for the
cells and does not influence endogenous gene expression. Despite this, a number of side effects have been
reported in in vitro and in vivo studies, suggesting that
GFP can affect cell physiology and proper cellular functions. Although to date, the mechanism by which GFP
provokes some described defects remains undefined, the
subtle consequences of its expression are of considerable interest to a wide range of biologists.
A series of GFP-tagged variants of myosin VI, in which
particular sequences were deleted or altered, have been
extensively used in our research on the role of myosin
VI during the last step of Drosophila spermatogenesis,
called spermatid individualization (Isaji et al. 2011). In
these experiments males expressing full-length GFP-myosin VI transgene (GFP-M6FL) were able to complete
individualization to produce motile sperm and served as
a positive control for different myosin VI mutant variations. To determine whether GFP-M6FL expression
causes any possible side effects during Drosophila sperm
development, we performed electron microscopy on
cross sections of testes dissected from wild-type males
(WT) and GFP-M6FL males (expressing only transgene
or expressing transgene in the presence of endogenous
myosin VI). Our comparative analysis showed that
GFP-M6FL expression can induce subtle ultrastructural
changes in maturing spermatids, particularly when no
endogenous myosin VI is present in the testes. The nature of these differences seems minor and males were
fertile, suggesting there is no serious effect of GFPM6FL expression on maturing spermatids.
In conclusion, our data confirm the idea that GFP is nontoxic to the cells and does not affect proper cellular function. However, possible side effects of GFP-transgene expression should be kept in mind when using this marker to
track cells because the induced fine changes in cellular organization may confound interpretation of functional data. The adult bone marrow (BM) harbors Sca-1+/Lin-/CD45population enriched in Oct-4+/ Nanog+ very small embryonic-like stem cells (VSELs), which may express early
differentiation markers of several lineages including vascular cells. VSELs may i) be mobilized in tissue injury and ii)
participate in tissue regeneration including in heart repair
in mice. However, their activation and proliferation during
such injury/ repair events in vivo have not been reported.
In this study, we examined if acute ischemic limb injury
(ILI) may stimulate both proliferation of quiescent VSELs
in BM and their mobilization into peripheral blood (PB).
Thus, 4-6 week old C57BL/6 mice underwent a hind limb
ischemia by permanent proximal femoral artery occlusion.
Mice were administrated with bromodeoxyuridine (BrdU)
and scarified at 2, 7, 14 and 28 days following ischemia. PB
and BM from non- and ischemic limbs were collected from
individual animals. Healthy non-ischemic mice were used as
control groups in each time point. The presence of proliferating (BrdU+) VSELs (Sca-1+/Lin-/CD45-), endothelial
progenitor cells (EPCs; Flk-1+/Sca-1+/Lin-/CD45-dim)
and hematopoietic stem/progenitor cells (HSPCs; Sca-1+/
Lin-/ CD45+) in PB and BM was evaluated by multiparameter flow cytometry and ImageStream system. The expression of genes related to the presence of VSEL (Oct-4,
Nanog) and EPC (Tie2, VE-cadherin) fractions was examined by real-time RT-PCR. Moreover, we examined the
change in expression of 53 angiogenesis-related proteins in
plasma at indicated time points following injury.
We found that the number of non-proliferating VSELs was
significantly increased in BM of ischemic mice at 7d post
injury. Similar results were obtained for EPCs. Increased
number of BrdU+ VSELs in BM and circulating in PB of
the injured animals was accompanied with change in expression of genes guiding their proliferation (Igf2, H19).
We defined the most important angiogenesis-related proteins which may stimulate proliferation and mobilization
of VSELs.
The data indicates the ischemic injury may recruit the
normally quiescent pluripotent stem cell pools from BM
niche to enhance endogenous mechanisms of tissue repair
including stem/ progenitor cell- dependent angiogenesis
which we currently further investigate. We conclude that
acute tissue injury such as limb ischemia may provide stimulatory agents to activate proliferation of quiescence VSELs residing in BM followed by their mobilization into PB.
Acknowledgments:
This work was supported by Polish Ministry of Science and Higher Education grant NN303 816240 (to M.L.).
References:
Isaji M et al (2011) PLoS ONE 6: e22755.

Lectures L5.1 L5.2 Session 5. Stem Cells and Regenerative Medicine

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