Læknablaðið : fylgirit - 03.01.2017, Blaðsíða 45

X V I I I V Í S I N D A R Á Ð S T E F N A H Í
F Y L G I R I T 9 1
LÆKNAblaðið/Fylgirit 91 2017/103 45
CRISPR DNA-editing to engineer the T-risk allele in heterozygous or
homozygous forms into the cell lines.
Results: Using a bioinformatics approach we have identified several
transcription factor binding sites at the polymorphic promoter site, inclu-
ding a possible CEBP-beta site. Furthermore, we have seen a marked
increase in MUC5B expression after induction with interleukin(IL)-13,
and under these conditions there is also a marked increase in CEBP-beta
expression. BCi_NS1.1 cells transduced with the T-risk allele had a 33,6%
increase in luciferase signal, when compared to wild-type. This signal was
further amplified to 54,21% after transient overexpression of CEBP-beta.
Conclusions: Our results indicate that the T allele has a stimulatory effect
on MUC5B expression, that could be mediated by C/EBP-beta.
E 127 Pathogen inactivated platelet lysates boost osteogenic and
chondrogenic differentiation of MSC and mesenchymal progenitor
cells
Ólafur E. Sigurjónsson1, Sandra Jonsdottir-Buch2, Kristbjörg Gunnarsdóttir2
1Surgical Services, The Icelandic Blood Bank, Landspitali Hospital, 2Department of Medicine,
University of Iceland
oes@hi.is
Introduction: MSC are interesting due to their multipotent differentiation
and immunomodulatory abilities. Embryonic derived mesenchymal
progenitor cells (hES-MP) have the same characteristics as MSC. Addition
of animal-serum is currently required for the survival and growth of
hES-MP and MSC. Using animal-serum to grow cells intended for human
medical therapy is not safe and serum-free alternatives need to be found.
Here we show that using platelet lysates made from pathogen inactivated
platelets (hPL-PI) during differentiation of MSC and hES-MP supports
both chondrogenesis and osteogenesis allowing serum-free growth
conditions from expansion throughout differentiation.
Methods: MSC and hES-MP002.5 were grown and differentiated towards
chondrogenic and osteogenic lineages in the presence of fetal bovine
serum (FBS) or hPL-PI. Chondrogenic differentiation was evaluated
over five weeks and histology and gene expression evaluated weekly.
Osteogenic differentiation was performed over four weeks with alkaline
phosphatase activity, mineralization and gene expression evaluated
weekly.
Results: Histology resembling articular cartilage was observed for MSC
and hES-MP at the end of chondrogenic differentiation when differ-
entiated in the presence of hPL-PI. SOX9, the master transcription factor
of chondrogenesis, was significantly upregulated in cell differentiated in
hPL-PI compared to FBS. During osteogenic differentiation the activity of
alkaline phosphatase, amount of calcium deposits and gene expression
was significantly higher in MSC and hES-MP differentiated in hPL-PI
compared to FBS.
Conclusions: Using hPL-PI based differentiation media encourages
osteogenic and chondrogenic differentiation of MSC and hES-MP to a
greater degree than FBS based media.
E 128 Embryonic stem cell derived mesenchymal progenitors (hES-
MP) exhibit superior growth but inferior immunosuppression to MSC
Ólafur E. Sigurjónsson1, Sandra Jonsdottir-Buch2, Kristbjörg Gunnarsdóttir2
1Surgical Services, The Icelandic Blood Bank, Landspitali Hospital, Landspitalinn, 2Department
of Medicine, University of Iceland
oes@hi.is
Introduction: Mesenchymal stem cells (MSCs) are promising candidates
for cellular therapy due to their abilities to regenerate bone and cartilage
and their immunosuppressive characteristics. A problem regarding the
use of MSC in cellular therapy is the need for using fetal bovine serum
(FBS) during ex-vivo expansion of the cells prior to use. Lysates derived
from platelets have been suggested as an alternative, mainly due to the
abundance of various growth factors and cytokines that are found within
the granules of platelets. We have previously demonstrated that lysates
from expired platelet concentrates (hPLex) can be used for the expansion
and differentiation of human bone marrow derived MSC. Here we demon-
strate that platelet lysates produced from expired platelet concentrates can
be used successfully for the expansion and differentiation of hES-MP cells.
Methods: hES-MP cells were expanded in DMEM-F12 cell culture media
supplemented with either 10% FBS or 10% hPLex. hES-MP cells were
analyzed for expansion, expression of surface markers and osteogenic,
adipogenic and chondrogenic differentiation as well as their ability to
suppress immune-response in an MLR assay.
Results: When expanded with hPLex hES-MP cells maintained their mes-
enchymal characteristics had an equivalent growth and differentiation
features compared to cells expanded in FBS. However hES-MP cells
demonstrated superior growth to MSC while lacking the immunosupp-
ressive abilities.
Conclusion: Our findings suggest that platelet lysates produced from
expired platelet concentrates can be used as an alternative to fetal bovine
serum for the expansion of hES-MP cells.
E 129 Transciptional control of pluripotent state transitions
Erna Magnúsdóttir, Kristján Hólm Grétarsson
Faculty of medicine, University of Iceland
erna@hi.is
Introduction: Mouse embryonic stem cells can recapitulate the
pluripotent state transitions occurring in the early embryo, where the
epiblast cells transition from naive to primed pluripotency. How this
transition is orchestrated is a key question in stem cell biology. We are
working on identifying transcriptional regulators that parttake in these
transitions.
Methods: We use mouse embryonic stem cells cultured in medium that
facilitates naive pluripotency and differentiate them with Activin and
FGF2 to induce primed pluripotency. We have generated loss of funct-
ion cells for transcriptional regulators, and assess their involvement in
the transition by comparing wild type control cells to the loss of function
cells during this transition. We monitor morphology, gene expression
and reporter activity.
Results: We have identified a transcriptional regulator that is key in
facilitating the transition from naive to primed pluripotency, and show
that in its absence the induction dynamics of primed pluripotency
markers is slowed down. We are in the process of testing lineage
commitment to the three germ layers as well as primordial germ cells
and assessing genome wide binding patterns of the factor.