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Mesenchymal progenitor cells in intramuscular connective tissue
development
Mesenchymal progenitor cells in intramuscular connective tissue
development
Min Du, Ph.D. Professor and Endowed Chair
Department of Animal SciencesWashington State University
Pullman, WA
Beef quality is mainly determined by marbling and tenderness.
Marbling is the primary criterion for grading beef carcasses.
Only carcasses with moderate to abundant marbling qualify for high quality grades.
Beef Quality: marbling and tenderness
Beef tenderness is determined by:
Myofibrillar effect: aging, stretching carcasses improves tenderness.
Background toughness: connective tissue, mainly collagen and its cross‐linking.
Both collagen content and cross‐linking increase as animals become older.
Question: How to reduce collagen content and cross‐linking in muscle?
Beef Quality: marbling and tenderness
Collagen content and cross‐linking, two critical control points:
Fibroblasts synthesize collagen and other fibrotic proteins in connective tissue.
Fibroblasts secrete enzymes catalyzing collagen cross‐linking.
Tenderness remains a top problem for beef
Collagen content
Collagen cross‐linking
How to reduce fibroblasts?
How to reduce fibroblasts?
Fibroblasts are derived from mesenchymal multipotent cells, primarily during the early developmental stage.
More importantly, fibroblasts and adipocytes share a common immediate progenitor cells, so called fibro/adipogenic cells (FAPs).
Tenderness remains a top problem for beef
: Why do some stem cells become myogenic cells, while others become fibro/adipogenic cells?
: Why do some progenitor cells become adipocytes, while others become fibrogenic cells?
Adipocytes
Fibroblasts
Satellite cells
Myocytes
Myogenic progenitor cells
Fibro/adipogenic progenitor cells
in fetuses
Stem cells in early embryos
Resident fibro/adipogenicprogenitor cells
In the stromal vascular fraction of mature muscle
1
2
1
2
The mechanisms regulating mesenchymal progenitor cell commitment to myogenesis, adipogenesis and fibrogenesis are poorly defined, especially in livestock.
Adipocytes
Fibroblasts
Satellite cells
Myocytes
Myogenic progenitor cells
Fibro/adipogenic progenitor cells
In fetuses
Stem cells in early embryos
Resident fibro/adipogenicprogenitor cells
In the stromal vascular fraction of mature muscle
1
2
Tumor necrosis factor (TGF) signaling pathway and Zfp423 transcription factor appear to have critical roles in determining progenitor commitments to either adipogenicor fibrogenic lineages.
Adipocytes
Fibroblasts
Satellite cells
Myocytes
Myogenic progenitor cells
Fibro/adipogenic progenitor cells
In fetuses
Stem cells in early embryos
Resident fibro/adipogenicprogenitor cells
In the stromal vascular fraction of mature muscle
1
2
Transforming growth factor (TGF)and fibrogenesis
TGF is a major signaling pathway promoting fibrogenesis.
TGF signaling is enhanced by inflammation and other factors.
Obesity is known to induce inflammation and TGFsignaling.
Fetal stage is critical for fibrogenesis.
AAAAA
Smad7CollagenCross‐linking enzymes
Smad 2
TGFRII
Kinase
TGFRI
Kinase
TGFRII
Kinase
TGFRI
Kinase
TGF
S465
S467
Smad 3
S422
S424
Smad 3
P
PP
Smad 3
P
PP
GST204
Objective: Thus, a maternal obesity sheep model was used to assess the role of TGF‐β signaling on fibrogenesis during early development.
Animals: Non‐pregnant ewes were assigned to a control diet (Con, fed 100% of NRCnutrient recommendations, n = 6) or obesogenicdiet (OB) fed 150% of NRC recommendations, n = 6) from 60 days before conception. Fetal semitendinosus (St) muscle was sampled at 135 days of gestation (term 148 days).
Methods: Histochemical analyses, Hydroxyproline assay, Real‐time PCR, Western blot analyses, Electrophoretic mobility shift assay (EMSA).
Experimental design
Huang et al., AJP‐ Endocrinology and Metabolism 298:1254‐1260, 2010
0
0.5
1
1.5
Con OB
Arb
itary
Uni
t *
Xu et al., Endocrinology, 2010, 151: 380.
Enhanced tumor necrosis factor (TNF)expression in OB fetal muscle
TNF: A marker of systemic inflammation.NF‐B: A major inflammatory signaling.
0.2
0.6
1
1.4
1.8 *
NF-B p65
OB
GAPDH
Con
00.5
11.5
22.5
33.5
Con OB
TGFβ
Prec
urso
r
Tubulin
TGFβ Precursor
*
Con OB Con OB
Huang et al., AJP‐ Endocrinology and Metabolism 298:1254‐1260, 2010
Transforming growth factor (TGF) content increased in
OB fetal muscle
AAAAA
Smad7CollagenCross‐linking enzymes
Smad 2
TGFRII
Kinase
TGFRI
Kinase
TGFRII
Kinase
TGFRI
Kinase
TGF
S465
S467
Smad 3
S422
S424
Smad 3
P
PP
Smad 3
P
PP
GST204
Smad binding element (SBE)
Huang et al., AJP‐ Endocrinology and Metabolism 298:1254‐1260, 2010
Binding to Smad responsive element was increased in OB fetal muscle
Shift band
Free probe
Con OB
0
0.5
1
1.5
2
2.5
Con OB
*
Rel
ativ
e SB
E sh
ift
P32 labeled Smad binding
elements
0123456
Con OB
Smad
7
*P < 0.05
Huang et al., AJP‐ Endocrinology and Metabolism 298:1254‐1260, 2010
Expression of Smad7 was enhanced in OB
fetal muscle
AAAAA
Smad7CollagenCross‐linking enzymes
Smad 2
TGFRII
Kinase
TGFRI
Kinase
TGFRII
Kinase
TGFRI
Kinase
TGF
S465
S467
Smad 3
S422
S424
Smad 3
P
PP
Smad 3
P
PP
GST204
0.0
1.0
2.0
3.0
4.0
Con OB
Fibr
onec
tin
*P < 0.05
00.5
11.5
22.5
33.5
Con OB
Proc
olla
gen
Type
I
&P = 0.08
Procollagen: Precursor of collagen.Fibronectin: Extracellular matrix glycoprotein.
Huang et al., AJP‐ Endocrinology and Metabolism 298:1254‐1260, 2010
Expression of TGF target genes was enhanced (Connective tissue content)
0
1
2
3
4
Con OB
LH2b
*P < 0.05
0123456
Con OB
Lysy
loxi
dase
*P < 0.05
00.5
11.5
22.5
Con OB
P4H
A
&P = 0.05
P4HA: prolyl 4‐hydroxylase, formation of hydroxyproline.LH2b: lysyl hydroxylase‐2b, collagen cross‐linking.Lysyl oxidase: amine oxidase, collagen cross‐linking.
Huang et al., AJP‐ Endocrinology and Metabolism 298:1254‐1260, 2010
Expression of TGF target genes was enhanced (Collagen cross‐linking)
40
80
120
160
Con OB
0
10
20
30
40
Con OB
Col
lage
n ar
ea/v
iew
ar
ea (%
)
*
Col
lage
n/dr
y m
uscl
e w
eigh
t (m
g/g)
CON
OB
x100 x400
Huang et al., AJP‐ Endocrinology and Metabolism 298:1254‐1260, 2010
*
Transforming growth factor (TGF)and fibrogenesis
Sheep fetal muscle at 135 dG when skeletal muscle matures (term day 148 gestation).
Con OB
Xu et al., Endocrinology, 2010, 151: 380.
It appears that the fibro/adipogenic pathway was enhanced in fetal muscle due to obesity and over‐nutrition in early development – Control point 1.
Adipocytes
Fibroblasts
Satellite cells
Myocytes
Myogenic progenitor cells
Fibro/adipogenic progenitor cells
In fetuses
Stem cells in early embryos
Resident fibro/adipogenicprogenitor cells
In the stromal vascular fraction of mature muscle
1
2
To further study mechanisms regulating lineage commitments of mesenchymal stem cells and progenitor cells, we used Wagyu and Angus cattle.
Adipocytes
Fibroblasts
Satellite cells
Myocytes
Myogenic progenitor cells
Fibro/adipogenic progenitor cells
In fetuses
Stem cells in early embryos
Resident fibro/adipogenicprogenitor cells
In the stromal vascular fraction of mature muscle
1
2
Wagyu cattle are known for its extremely high marbling
Wagyu cattle are known for their extremely high marbling.
Due to their similarities in growth characteristics, but sufficient difference in marbling, Wagyu and Angus cattle are frequently compared.
We sampled the Sternomandibularismuscle for analyses.
0.00.51.01.52.02.5
CEBPβ
P = 0.2864
Arb
itrar
y un
its
0.01.02.03.04.05.0
PPARγ
Arb
itrar
y un
its *
0.00.51.01.52.02.5
Zfp423
Arb
itrar
y un
its *
0.0
1.0
2.0
3.0
4.0
Intramuscular fat(IMF)
*
% o
f eth
er e
xtra
ct
Wagyu muscle have enhanced adipogenesis
0.0
1.0
2.0
3.0
4.0
CEBPα
*
Arb
itrar
y un
its
Duarte et al., J. Anim. Sci., 2013, 91: 2938
0.00.51.01.52.02.5
FGF-2
Arb
itrar
y un
its
*
0.00.51.01.52.0
FGFr
Arb
itrar
y un
its
FGFR
*
0.0
1.0
2.0
3.0
FIBRONECTIN
Arb
itrar
y un
its
Fibronectin
*
0.0
1.0
2.0
3.0
4.0
TGFβ
Arb
itrar
y un
its *
Wagyu cattle have higher fibrogenesis
Duarte et al., J. Anim. Sci., 2013, 91: 2938
0.0
1.0
2.0
3.0
4.0
COL I COL IIICol
lage
n m
RN
A ex
pres
sion
(arb
itrar
y un
its)
Collagen I Collagen III
*
*
0.0
1.0
2.0
3.0
4.0
Collagen content
*
mg/
g of
mus
cle
Wagyu cattle are higher collagen content
Duarte et al., J. Anim. Sci., 2013, 91: 2938
100x 200x 400x
Angus
Wagyu
Wagyu cattle are higher collagen content
Duarte et al., J. Anim. Sci., 2013, 91: 2938
Wagyu
Angus
0
50
100
150
20 45 70 95 120
Diameter (µm)
Freq
uenc
y
020406080
100
Breeds
*
Dia
met
er
(µm
)
Angus Wagyu
Duarte et al., J. Anim. Sci., 2013, 91: 2938
Wagyu have decreased myogenic pathway
Attenuated fetal myogenesis forms less muscle fibers.
The larger muscle fiber diameter in Wagyu despite less muscle mass shows less muscle fiber numbers, indicating attenuated myogenesis during early development.
Wagyu
Angus
It appears that both fibro/adipogenic pathway (1st question) and adipogenic differentiation (2nd question) are enhanced in Wagyu, which we are exploring.
Our current studies focus on Zfp423.
Adipocytes
Fibroblasts
Satellite cells
Myocytes
Myogenic progenitor cells
Fibro/adipogenic progenitor cells
In fetuses
Stem cells in early embryos
Resident fibro/adipogenicprogenitor cells
In the stromal vascular fraction of mature muscle
1
2
Zfp423 in adipogenesis of fibro/adipogenicprogenitor cells
Key question 2: what determines the lineage commitment of fibro/adipogenic cells (FAPs)?
Zinc finger protein (Zfp) 423 is a newly identified transcription factor regulating adipogeniccommitment of FAPs, which induces adipogenicdifferentiation and reduces fibrogenesis.
AdipocytesFAPs
Zfp423 PPAR
Pre‐Adipocytes
Adipogeniccommitment
Adipogenicdifferentiation
What is Zfp423?
We hypothesized that Zfp423 is critical for adipogenic differentiation of intramuscular fibro/adipogenic progenitor cells.
Zfp423 is correlated with enhanced adipogenesis and reduced fibrogenesis in beef cattle.
Animal:
The Sternocleidomastoid muscle was sampled from the carcass of an Angus heifer (20 months of age) immediately after slaughter.
Stromal vascular cells were separated, immortalized by over‐expression of telomerase, and cloned.
Three clones with high adipogenic and low adipogenicpotential respectively were selected.
Methods
0
0.5
1
1.5
2
2.5
Highadipogenic
Low adipogenic
*
High adipogenic
Low adipogenic
A
B
C
D
Arbitrary un
its
Zfp423 in adipogenesis of fibro/adipogenicprogenitor cells
Huang et al., PLOS one, 2012, 7: e47496
0.0
1.0
2.0
3.0
4.0
5.0
Zfp423 PPARr C/EBPa C/EBPb Fibronectin TGFb CollagenI CollagenIII
0.0
3.0
6.0
9.0
Zfp423 PPARr C/EBPa C/EBPb Fibronectin TGFb CollagenI CollagenIII
Before differentiation
Adipogenic differentiation for 13 days
**
*
**
&&
& &
Arbitrary un
itsArbitrary un
its
C/EBPαPPARγ C/EBP
C/EBPαPPARγ C/EBP
TGF‐
TGF‐
mRNA expression of Zfp423 and other genes
Low adipogenicHigh adipogenic
*P < 0.05; &P < 0.10.
Huang et al., PLOS one, 2012, 7: e47496
0
0.4
0.8
1.2
1.6
2
Low adipogenic High adipogenic
After adipogenic differentiationBefore adipogenic differentiation
&
0.0
1.0
2.0
3.0
4.0
Low adipogenic High adipogenic
*
High adipogenicLow adipogenic
TGF‐
Tubulin
High adipogenicLow adipogenic
TGF‐
Tubulin
Arbitrary un
its
Arbitrary un
its
TGF signaling was higher in Low Adipogenic Cells
Huang et al., PLOS one, 2012, 7: e47496
0
0.5
1
1.5
2
2.5
3
3.5
4
Zfp423 C/EBPa C/EBPb PPARr Fibronectin TGFb
Adipogenic Non‐adipogenic
NA Zfp423 transfection NA GFP transfection
aa
bba abbc
ca aab b
a
a
bcc
ab b
ab ab b
ab
Arbitrary un
its
C/EBP C/EBP PPAR TGF‐
Zfp423 over‐expression in low adipogenic cells enhances their adipogenic differentiation
(Bars marked with different letter differ, n =3.)Huang et al., PLOS one, 2012, 7: e47496
TGF‐
Zfp423 over‐expression in low adipogenic cells enhances their adipogenic differentiation
(Bars marked with different letter differ, n =3.)
Huang et al., PLOS one, 2012, 7: e47496
High adipogenic Low adipogenic Low adipogenic + Zfp423 Low adipogenic + eGFP
Zfp423 in adipogenesis of fibro/adipogenicprogenitor cells
High adipogenicLow adipogenic High adipogenic + shZfp423
High adipogenic + GFP
Huang et al., PLOS one, 2012, 7: e47496
Both high and low adipogenic cells are from the same bovine animal.
They have identical genetic composition.
The difference in their adipogenic and fibrogenicdifferentiation should be due to epigenetic modifications.
Because these cells have been immortalized and cloned, only stable epigenetic modifications, or DNA methylation, is expected to maintain.
Why does Zfp423 express at a higher level in high adipogenic cells?
0
1
2
3
4
5
TGF‐b Zfp423
Low adipogenic
High adipogenic
*
*
Arbitrary un
its
TGF‐
Zfp423 promotor contains rich GC sites, and higher methylation in low compared to high adipogenic cells
ATG
Methylated DNA immunoprecipitation (MeDIP) was used to measure the DNA methylation of Zfp423 promoter.
Huang et al., PLOS one, 2012, 7: e47496
Zfp423 in adipogenesis of fibro/adipogenicprogenitor cells
Why do TGF and Zfp423 expression differ between low and high adipogenic cells?
DNA methylation is a stable epigenetic modification which inhibits gene expression, determining cell phenotypes.
TGF DNA methylation was higher, while Zfp423 DNA methylation was lower in low adipogenic cells, consistent with higher adipogenic and low fibrogenic differentiation of FAPs.
Huang et al., PLOS one, 2012, 7: e47496
What regulates Zfp423 expression?
We hypothesized that epigenetic modifications have key roles in regulating Zfp423 expression.
Epigenetic modifications are mainly referring to DNA methylation and histone modifications.
Histone modifications include histone methylation and acetylation, and others.
What regulates Zfp423 expression?
Polycomb repressive complex 2 (PGC2) catalyzes repressive histone modifications, H3K27me3.
Trithorax group proteins (TrxG) catalyze permissive histone modifications, H3K4me3.
Both H3K27me3 and H3K4me3 co‐exist in key developmental genes, forming “bivalent” status.
Lack of stimulation, repressive histone modifications convert to DNA methylation for permanent silencing.
Sect 1 Sect 2Sect 3 Sect 4
B
A
Yang et al., Diabetes, 2013, 62:3727
Zfp423 promoter has a “bivalent” status
Maternal obesity enhances Zfp423 expression in fetal tissue via inducing epigenetic changes
To test the role of epigenetic changes in the regulation of Zfp423 expression, we used a diet‐induced obesity pregnant mouse model.
Female mice were fed either a control diet (Con) or an obesogenic diet (OB) for two months to induce obesity.
Fetal mice at E14.5 were collected for analyses, when early adipose development has initiated.
Con
OB
Sec1 Sec 2 Sec3 Sec4
CON vs. OB: 30.0% vs. 16.9% 38.0% vs. 21.0% 4.4% vs. 2.1% 0.4% vs. 0.0%
Yang et al., Diabetes, 2013, 62:3727
DNA methylation was lower in Zfp423 of fetal tissue of obese mothers
0.00
2.00
4.00
6.00
8.00
10.00
Con OB
H3K27me3
Perc
ent o
f inp
ut (%
)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
Con OB
Percen
t of inp
ut (%
)
H3K4me3
*
H3K27me3 H3K4me3
*
IPInput
IgG
Con OB
IgGInput
IP
Yang et al., Diabetes, 2013, 62:3727
Inhibitory histone modification, H3K27me3 is lower, and permissive modification, H3K4me3, is higher in OB
Yang et al., Diabetes, 2013, 62:3727
Inhibitory histone modification, H3K27me3 is lower, and permissive modification, H3K4me3, is higher in OB
0
0.2
0.4
0.6
0.8
1
1.2
Con HFD
*
Zfp4
23 p
rote
in c
onte
nt(A
rbitr
ary
Uni
t)
OB
Con OB
Zfp423
β-Actin
H3K27me3 Methylated CpG Unmethylated CpG
Normal pregnancy
PRC2
Zfp423 promoter
PRC2
Maternal obesity
PRC2PRC2
Zfp423 promoter
Acetylation
AC
ACACAC
Zfp423 Zfp423
Proposed mechanism linking maternal obesity to epigenetic modifications in the Zfp423 promoter
In summary, there are two major control points for reducing intramuscular fibrogenesis: Stem cells to either myogenic or FAP lineage. FAPs to either adipogenic or fibrogenic lineage.
Adipocytes
Fibroblasts
Satellite cells
Myocytes
Myogenic progenitor cells
Fibro/adipogenic progenitor cells
In fetuses
Stem cells in early embryos
Resident fibro/adipogenicprogenitor cells
In the stromal vascular fraction of mature muscle
1
2
Multipotentcells
Muscle cells
Adipocytes
Fibroblasts
Epigenetic changes in progenitor
cells
cell signaling pathways
Genetic, nutritional and other factors
Mechanisms regulating mesenchymalprogenitor cell differentiation
What controls mesenchymal progenitor cell differentiation?
Critical questions.
If we know these answers,
We can:
Improve lean/fat ratio and production efficiency.
Reduces connective deposition and increase tenderness of beef.
Enhance intramuscular adipogenesis and marbling.
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