apoptosis, therefore the expression level of c-MYC was almost
the same as that of the negative control. KLF4 might be
required to suppress p53 and c-Myc-induced apoptosis.
Fabp4
, which is expressed during terminal differentiation,
was not expressed in K- and M-transduced CMPs (Figure 4b).
This observation might be ascribed to the inability of those
CMPs to differentiate into mature lipid-laden adipocytes.
Furthermore, KM genes in CMPs resulted in a greater relative
area of Oil Red O-positive cells than that observed for the
transduction of OSKM, OKM and SKM (Figure 4c). These
results showed that the combination of
Klf4
and
c-Myc
is
indispensable for the differentiation of CMPs into adipocytes.
Klf4
and
c-Myc
do not induce adipogenic differentiation
of MSCs.
To test the ability of other cell types to differentiate
into adipocytes via KM transduction, we used the same
methods to induce adipocyte differentiation (Figure 1a).
KM genes did not increase the frequency of adipocyte
differentiation in 3T3-L1 preadipocytes (Figure 5a). All of the
adipogenic genes exhibited a similar expression pattern in
both non-treated and KM-transduced 3T3-L1 preadipocytes
(Figure 5b). Furthermore, we examined whether other mouse
multipotent MSCs derived from bone marrow (KUSA-A1,
KUM5 and KUM9 cells) could be induced to undergo
differentiation to adipocytes by KM transduction. Cells treated
Figure 1
OSKM-transduced CMPs differentiated into adipocytes. (
a
) Schematic representation of the adipocyte differentiation method. MC, medium change. Growth medium
indicates the basal medium for each cell line, and reprogramming medium indicates KO-DMEM-based medium. (
b
) Phase contrast microscope images. CMPs treated with OSKM
Sendai virus (CMP with OSKM) clearly accumulated large cytosolic lipid droplets at day 8. These droplets were stained with Oil Red O. Untreated CMPs (CMP cntrl) and those
treated with adipogenic stimulation cocktails (CMP with adipogenic cocktails) did not form cytosolic lipid droplets at day 8 and were not stained with Oil Red O. The white bar
indicates 50
μ
m. (
c
) qRT-PCR analysis of the expression of each gene in CMPs on each day. Individual RNA expression levels were normalized to
Gapdh
expression. Error bars
indicate S.E. (
n
=
3). * and ** indicate significant changes compared with untreated controls at day 8 (
P
o
0.05 and 0.01, respectively)
Cardiac adiposity is regulated by
Klf4
and
c-Myc
D Kami
et al
3
Cell Death and Disease
