Background: The
potential use of cell-based therapy is one of the most exciting advances in the
field of translational medicine to treat various pathological conditions that
have been untreatable up to now. Chronic lung disease is rapidly becoming the
primary cause of death in the US .
Currently, there is no intervention that can either arrest or cure evolving or
established chronic lung diseases. Knowledge of the intrinsic molecular
mechanisms that allow for resistance to inflammation and recruitment of stem
cells for repair would allow for the engineering of such cells for effective
treatment.
MSCs are bone marrow non-haematopoietic stem cells that are
multipotent and can differentiate into bone, cartilage and connective tissues.
Moreover MSCs present many advantages as facilities to culture or to transform
genetically. We have discovered that Parathyroid Hormone-related Protein
(PTHrP) and PPAR-r drive the key homeostatic lung epithelial-mesenchymal
interactions, resulting in the differentiation of alveolar interstitial
lipofibroblasts that are essential for normal lung development and its repair following
injury. In this paper, we try to isolate CD45 (-) CD73 (+) CD90 (+) MSCs from 6
weeks old rats and further induce such cells into adipogenic cells. Meanwhile,
we try to confirm our hypothesis that PTHrP and PPAR-γ related pathways are
involved in the MSCs differential procedure.
Methods: Femurs and tibias were aseptically harvested
from 6 weeks old Wistar rats. Whole bone marrow plugs were obtained by flushing
the bone marrow cavity with an 18-gauge needle set with a syringe filled with α-MEM
medium. Second passage of adherent MSCs were analyzed by flow cytometry after
staining with fluorochrome conjugated CD45, CD73 and CD90 antibodies. These
MSCs had been inducing with induction buffer (α-MEM enriched with 10% FBS, 10 μg/ml
insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine for 11
days. Oil Red O stain was processed to show the efficacy of adipocytic
induction. Total RNA was extracted with one step procedure, and RT-PCR was used
to probe PPAR-γ expression. Also, we investigated the muscular marker, α-SMA,
by Western blot hybridization.
Results: The
BM-derived MSCs expressed a set of specific markers that are well-known to
define MSCs, including 99.36% CD45 (-), 59.92 CD73 (+), and 91.1% CD90 (+).
Adipocytic differentiation of rat MSCs was induced under the conditions
described above. Morphologically, the spindle small MSCs changed into large
round cells with alveolus-like distribution.
Appearance of cell fat granules was observed after 11 days incubation.
More than 50% of the induced cells displayed Oil Red O+ lipid granules, and 500
nM PTHrP and 1 nM RGZ obviously increased the Oil Red O+ cells with more and
larger lipid granules. PPAR-γ, a marker of adipocylic differentian, was over
expressed after induction with/without enhancement (PTHrP or RGZ, or both). On
the other hand, the muscular marker, α-SMA had much lower expression after the
induction. The groups with PTHrP and/or RGZ had significant weaker α-SMA
expression, comparatively.
Conclusion:
Adherent MSCs isolated from 6 weeks old rat can be directionally induced into
adipocytic cells. PTHrP and PPAR-γ stimulator RGZ can enhance this kind of
differentiation.
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