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Cardiac mesenchymal progenitors differentiate into
adipocytes via

Klf4

and

c-Myc

D Kami

1

, T Kitani

2

, T Kawasaki

2

and S Gojo*

,1

Direct reprogramming of differentiated cells to pluripotent stem cells has great potential to improve our understanding of
developmental biology and disorders such as cancers, and has implications for regenerative medicine. In general, the effects of
transcription factors (TFs) that are transduced into cells can be influenced by pre-existing transcriptional networks and epigenetic
modifications. However, previous work has identified four key TFs,

Oct4, Sox2, Klf4

and

c-Myc

, which can reprogram various

differentiated cells to generate induced pluripotent stem cells. Here, we show that in the heart, the transduction of cardiac
mesenchymal progenitors (CMPs) with Klf4 and c-Myc (KM) was sufficient to drive the differentiation of these cells into adipocytes
without the use of adipogenic stimulation cocktail, that is, insulin, 3-isobutyl-1-methylxanthine (IBMX) and dexamethasone.
KM-transduced CMPs exhibited a gradually increased expression of adipogenic-related genes, such as

C/Ebp

α

,

Ppar

γ

and

Fabp4

,

activation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway, inactivation of the cell cycle-related pathway
and formation of cytoplasmic lipid droplets within 10 days. In contrast, NIH3T3 fibroblasts, 3T3-L1 preadipocytes, and bone
marrow-derived mesenchymal stem cells transduced with KM did not differentiate into adipocytes. Both

in vitro

and

in vivo

cardiac

ischemia reperfusion injury models demonstrated that the expression of KM genes sharply increased following a reperfusion
insult. These results suggest that ectopic adipose tissue formation in the heart following myocardial infarction results from CMPs
that express KM following a stress response.

Cell Death and Disease

(2016)

7,

e2190; doi:10.1038/cddis.2016.31; published online 14 April 2016

Adipocyte differentiation, that is, adipogenesis, has been
extensively investigated, and its regulation via transcriptional
cascades has been described for

in vitro

model systems.

1

The

adipogenic transcriptional cascade consists of two waves. The
first wave converges at the CCAAT/enhancer-binding protein
(C/Ebp)

β

/

γ

, which induces the second wave consisting of

nuclear receptor peroxisome proliferator-activated receptor
(Ppar)

γ

and C/EBP

α

activity. In addition,

c-Myc

is periodically

expressed during the early phase of adipogenesis.

2

Krüppel-

like factor (

Klf

) family members include both repressors and

activators of adipogenesis, and are activated during the first
wave.

3

KLF4 and c-MYC (KM) coordinately bind the promoters

of genes that are activated during the reprogramming of
differentiated cells to pluripotency.

4

Whether KM work together

in adipogenesis has not been examined.

Mesenchymal stem cells (MSCs) are multipotent cells with

a capacity to differentiate to mesodermal lineages and show
a vigorous proliferation capacity under conventional culture
conditions.

5

The criteria for identifying MSCs include adher-

ence to a plastic dish, a characteristic surface profile and

differentiation capacity

in vitro

.

6

Although most prior reports

have identified bone marrow as the origin for MSCs, other
organs including adipose tissue

7

and the heart

8,9

also harbor

fibroblasts that fulfill the criteria for MSCs. MSCs derived from
different organs demonstrate varying capacities for proliferation
and differentiation.

10

Although several reports have demon-

strated adipose tissue formation in the myocardium following
reperfusion therapy for ischemic heart diseases,

11

14

it is

unclear how fat depositions in the heart are generated.

Direct reprogramming of differentiated cells using specific

transcription factors (TFs) opens the door to understanding
the mechanisms underlying development and the pathogen-
esis of various disorders, and has applications in regenerative
medicine.

15,16

Transdifferentiation

or

direct

conversion,

which occurs when a differentiated cell type is reprogrammed
to another cell type, could be implemented via the same
strategy of using a set of TFs to generate cardiomyocytes,
neurons and so on.

17,18

Moreover, there are similarities and

overlaps between the pathways for the generation of induced
pluripotent stem cells (iPSCs) and tumorigenesis, such as a

1

Department of Regenerative Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan and

2

Department of Cardiovascular Medicine, Graduate School of Medical

Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
*Corresponding author: S Gojo, Department of Regenerative Medicine, Kyoto Prefectural University of Medicine, 465 Kajii cho, Kamigyo ku, Kyoto 602-8566, Japan.
Tel: +81 75 251 5752; Fax: +81 75 251 5910; E-mail: [email protected]

Received 09.7.15; revised 19.1.16; accepted 20.1.16; Edited by D Aberdam

Abbreviations:

C/EBP, CCAAT/enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; Klf, Krüppel-like factor; KM, KLF4 and c-MYC; MSC,

mesenchymal stem cell; TF, transcription factor; iPSC, induced pluripotent stem cell; OSKM,

Oct4, Sox2, Klf4

and

c-Myc

; CMP, cardiac mesenchymal progenitor; IRI,

ischemic reperfusion injury; MI, myocardial infarction; qRT-PCR, quantitative reverse transcription polymerase chain reaction; PCA, principal component analysis; PC,
principal component; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; LAD, left anterior descending artery; AON, area of necrosis; AAR, area
at risk; RA, remote area; ROS, reactive oxygen species; LV, left ventricular; RAAS, renin

angiotensin

aldosterone system; NRX, nucleoredoxin; Dvl, dishevelled; bFGF,

basic fibroblast growth factor; IBMX, 3-isobutyl-1-methylxanthine; DMEM, Dulbecco's modified Eagle

s medium; FBS, fetal bovine serum; MEM, minimum essential media;

KO-DMEM, knockout DMEM; PFA, paraformaldehyde; NIA array analysis, National Institute on Aging array analysis; DAVID, Database for Annotation, Visualization and
Integrated Discovery; GEO, Gene Expression Omnibus; TTC, 2,3,5-triphenyltetrazolium chloride

Citation: Cell Death and Disease (2016) 7, e2190; doi:10.1038/cddis.2016.31

&

2016 Macmillan Publishers Limited All rights reserved 2041-4889/16

www.nature.com/cddis

Содержание KTD50

Страница 1: ...onditions 5 The criteria for identifying MSCs include adher ence to a plastic dish a characteristic surface profile and differentiation capacity in vitro 6 Although most prior reports have identified bone marrow as the origin for MSCs other organs including adipose tissue7 and the heart8 9 also harbor fibroblasts that fulfill the criteria for MSCs MSCs derived from different organs demonstrate var...

Страница 2: ...microarray chip and the NIA Array Analysis website 22 Based on hierarchical clustering analysis of gene expression OSKM CMPs could be clearly discriminated from CMP controls Figure 3a In addition principal component analysis PCA of gene expression showed that the OSKM CMPs were different from the CMP controls and gradually shifted from right to left on the PC1 axis in a time dependent manner Figur...

Страница 3: ... 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 me...

Страница 4: ...1 were examined The expression of both c Fos and c Jun drastically and temporarily increased just after exposure to normoxic conditions Moreover we determined that KM was involved in in vivo murine cardiac IRI Figures 6d f Injured murine ventricles acutely and temporarily expressed Klf4 and c Myc in IRI similar to the pattern observed for in vitro IRI The expression levels of both c Fos and c Jun ...

Страница 5: ...sion of the first wave of TFs in AON was significantly higher than that in RA and the gene expression of the second wave of TFs was not significantly different among the three areas The surrogate TFs c Fos and c Jun showed significantly increased expres sion in AON and AAR compared with that in RA which was the same tendency as that observed for Klf4 c Myc C Ebpβ and C Ebpδ suggesting that adipoge...

Страница 6: ...anges compared with KM treated CMPs white box Po0 05 and 0 01 respectively c Calculation of Oil Red O staining area Each well image was captured using a Keyence BZ X700 digital microscope The black bar indicates 5 mm left The graph shows the percentages of the total area that were positive for Oil Red O staining right Error bars indicate S E and indicate significant changes Po0 05 and 0 01 respect...

Страница 7: ... S E n 3 and indicate significant changes compared with KM treated 3T3 L1 cells at day 8 Po0 05 and 0 01 respectively c Phase contrast microscope images MSCs derived from mouse bone marrow were transduced with KM Sendai virus One day after infection cells were cultured in reprogramming medium for 8 days At day 8 cells were fixed and stained with Oil Red O The white bar indicates 50 μm Cntrl indica...

Страница 8: ...onditions in a hypoxic chamber and normoxia indicates 21 O2 5 CO2 conditions b Phase contrast microscope images of CMPs under hypoxic conditions c qRT PCR analysis of the expression of each gene in CMPs Error bars indicate S E n 3 and indicate significant changes compared with CMPs at 0 h white box Po0 05 and 0 01 respectively H indicates hypoxia condition N indicates hormoxia condition d Schemati...

Страница 9: ...tissue growth owing to the post mitotic nature of mature adipocytes In adipose tissue resident MSCs are considered to be a major source for adipocyte generation 36 Some studies have reported in vivo adipocyte differentiation from MSCs which expressed similar cell surface antigens to those expressed by the CMPs in this study 44 45 Recently myocardium derived stem progenitor cells such as cardiac st...

Страница 10: ...hcare UK Buckinghamshire England The adipogenic stimulation cocktail ingredients insulin IBMX and dexamethasone were purchased from Sigma Aldrich St Louis MO USA Cell preparation Experimental procedures and protocols were approved by the Animal Experiment Ethics Committee of the Kyoto Prefectural University of Medicine Murine CMPs were isolated from wild type C57BL 6 mouse hearts 10 to 16 week old...

Страница 11: ...s o0 05 were considered significant Conflict of Interest The authors declare no conflict of interest Acknowledgements We would like to express our sincere thanks to Toyoda Masashi Tokyo Metropolitan Institute of Gerontology for helpful discussions regarding the results presented in the manuscript This study was supported by a Grant in Aid for Exploratory Research from JSPS KAKENHI 24659594 1 Lefte...

Страница 12: ...y cardiosphere derived cells for heart regeneration after myocardial infarction CADUCEUS a prospective randomised phase 1 trial Lancet 2012 379 895 904 48 Smits AM van Vliet P Metz CH Korfage T Sluijter JP Doevendans PA et al Human cardiomyocyte progenitor cells differentiate into functional mature cardiomyocytes an in vitro model for studying human cardiac physiology and pathophysiology Nat Proto...

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