Reprogramming of mesenchymal stem cells and adult fibroblasts following nuclear transfer in rabbits

The main aim of this thesis was to find out which donor cells would
be most suitable for production of cloned rabbits with a targeted
modification of their genome, and their corresponding patterns of
reprogramming hetero- and euchromatic histone modifications
(H3K4m2/3 and H3K27m3). For these purposes, we carried out NT with
rabbit mesenchymal stem cells (MSCs) and adult fibroblasts (RAFs).
As totally 13 cell lines of MSCs have been tested, they didn’t show
stably higher development potential than RAFs, even though the
blastocyst rate of embryos cloned from MSC A/B reached to 76%.
Finally two cloned rabbits were produced from meschnymal stem
cells. H3K27m3 was undetectable in all stages of nuclear transfer
embryos except for one cell stage and blastocysts. It seems that
H3K27m3 is faithfully reprogrammed in transferred nuclei of all
donor cell types, with minor differences in zygotes and
blastocysts. Strong signals for H3K4m2/3 were detected at the one
to two-cell stages of in vivo embryos with a slight decrease at the
4-cell stage, followed by a more drastic decrease at the 8-cell
stage, where the signal minimum was reached. In 16-cell embryos
signals slightly increased and then reached in morulae and
blastocysts the levels observed in one-two cell embryos. In all
types of nuclear transfer embryos fluorescence intensity ratios
differed from that of in vivo embryos. The minimum was not reached
at the 8-cell stage but at the 4-cell stage. Reprogramming of
H3K4m2/3 modification occurred quite differently with either type
of cells irrespective of the cell origin or type and no close
similarities in the patterns of this reprogramming was observed
between in vivo and nuclear transfer embryos. Embryos cloned from
MSC A/B differed from all others in the way that H3K4m2/3 was
generally lower and remained in the range of the donor cells. This
suggests that reprogramming of H3K4m2/3 modification is more
aberrant with MSC A/B cells than with any other cell type used in
this study. However, with MSC A/B cells, a significantly higher or
similar proportion of cloned embryos developed to blastocysts
indicating that reprogramming of H3K4m2/3 modification does not
correlate with developmental potential of donor cells. In
conclusion, our study provides evidence that histone modifications
for heterochromatin are faithfully reprogrammed during NT of rabbit
somatic cells, while patterns of epigenetic reprogramming of
euchromatic histone modifications differ between individual cell
lines irrespective of their origin or type and are not correlated
with their developmental potential. Although MSCs were not superior
to RAFs in respect to the criteria tested in our study they may be
suitable nuclear donors for generating transgenic cloned rabbits
due to their high developmental plasticity.