a CCE time course

0

0.025

0.05

0 2 3 4 5 7 10 Embryo10.5EB Day

Pax

3

0.0021

0.0042

00 2 3 4 5 7 10 Embryo

10.5EB Day

Myf

5

0.06

0.12

00 2 3 4 5 7 10 Embryo

10.5EB Day

Myo

geni

n

0.0045

0.009

00 2 3 4 5 7 10 Embryo

10.5EB Day

Myo

D

b J1 time course

0.025

0.05

00 2 3 4 5 6 7 10 Embryo

10.5EB day

Pax

3

0.005

0.01

00 2 3 4 5 6 7 10 Embryo

10.5EB day

Myo

D

0.0018

0.0036

00 2 3 4 5 6 7 10 Embryo

10.5EB day

Myf

5

0.06

0.12

00 2 3 4 5 6 7 10 Embryo

10.5EB day

Myo

geni

n

Functional Skeletal Muscle Regeneration from Differentiating Embryonic Stem Cells

Radbod Darabi1, Kimberly Gehlbach1, Robert M. Bachoo2, Shwetha Kamath1, Mitsujiro Osawa1, Kristine E. Kamm3, Michael Kyba1, and Rita C. R. Perlingeiro1

Supplementary Figure 1

Supplementary Figure 1. Expression of myogenic regulators during EB development.(a–b) Expression in additional wild-type control ES cells. RNA was isolated from CCE (a) and J1 (b) ES cells (day 0) and EBs differentiated for 2, 3, 4, 5, 6, 7, and 10 days, and analyzed for the expression of specific myogenic markers by real time RT-PCR. (c) Dox has no effect on myogenic gene expression. Relative levels of gene expression in monolayer outgrowths derived from A2lox control EBs (days 3, 4, 5, and 6), grown entirely in the presence or absence of dox. EBs were harvested at the time indicated on the x axis and RT-PCR was performed on derivative monolayer cultures seven days later. Transcripts are normalized to GAPDH. Day 10.5 pc embryos were used as reference.

cA2lox (dox effect)

0.002

0.004

03 4 5 6 3 4 5 6 Embryo

10.5EB day

Myf

5

0.02

0.04

03 4 5 6 3 4 5 6 Embryo

10.5EB day

Pax

3

No dox Dox No dox Dox

0.005

0.01

03 4 5 6 3 4 5 6 Embryo

10.5EB day

Myo

D

0.04

0.08

0.12

03 4 5 6 3 4 5 6 Embryo

10.5EB day

Myo

geni

n

Supplementary Figure 1, cont.:

Muscle differentiation

CultureES cells

Lentiviralinfection

Sort GFP+ cells Into 96 well dish (1cell/well)a b

Supplementary Figure 2. Derivation of a GFP-expressing iPax3 ES cell clone. (a) iPax3 ES cells were transduced with a GFP lentiviral construct, and after two passages, the brightest GFP+ cells were single cell-sorted to generate GFP+ sub-clones. (b) Flow cytometry analysis of the selected clone of iPax3-GFP ES cells, in which EBs differentiated for 14 days show 100% GFP expression (thick line). The thin line represents fluorescence of the parental iPax3 negative control cells.(c) Cells from the same clone grown and differentiated as monolayer for 2 weeks show uniformly high GFP expression and muscle differentiation potential as demonstrated by MHC immunostaining. Cells are co-stained with DAPI (blue). Scale bar is 100 μm.

cDAPI GFP MHC Merge

EB day 14

100%

100 101 102 103 104

FITC-A

0

20

40

60

80

100

Cel

l cou

nts

No dox

Dox

Flk1-APC

PDGFαR-PE

EB d3 EB d4 EB d5

100 10 1 102 103 104100

101

102

103

104

9.55 9.06

4.0477.4

100 10 1 102 103 104100

101

102

103

104

14.2 9.89

3.7372.2

100 10 1 102 103 104100

101

102

103

104

25.5 16.1

15.443

100 10 1 102 103 104100

101

102

103

104

26.8 20.2

11.441.610 0 10 1 10 2 10 3 104

10 0

10 1

10 2

10 3

10 4

18.9 52.7

10.318.1

10 0 10 1 10 2 10 3 10410 0

10 1

10 2

10 3

10 4

17.7 46.5

14.421.5

PDGFαR- Flk-1–

Dox

PDGFαR– Flk-1+PDGFαR+Flk-1+

No dox

a

b

c

Supplementary Figure 3

0

0.08

0.16

Myo

geni

n

No dox Dox

0

0.02

0.04

DN

Flk-

1

PDG

FaR

DP

DN

Flk-

1

PDG

FaR

DP

Em

bryo

10.5

Myo

D

DN

Flk-

1

PDG

FaR

DP

DN

Flk-

1

PDG

FaR

DP

Em

bryo

10.5

Marker

PopulationMyf5(%)

MyoD(%)

MHC(%)

Unsorted 92.3 ± 2.1 32.8 ± 6.1 16.9 ± 2.4

PDGFαR– Flk-1– 86.4 ± 1.8 3.5 ± 1.1 1.2 ± 0.3

PDGFαR+Flk-1+ 90.9 ± 1.4 15.3 ± 1.3 10.5 ± 1.9

PDGFαR– Flk-1+ 95.5 ± 0.4 8.9 ± 2.5 3.9 ± 1.1

PDGFαR+Flk-1– 94.1 ± 2.7 79.3 ± 7.8 35.4 ± 5.9

d

Supplementary Figure 3, cont.:

Supplementary Figure 3. Acquisition of paraxial and myogenic markers in sorted fractions. (a) Dox has no effect on the differentiation of control ES cells. FACS profile of A2lox control ES cells during EB differentiation. Dox was added to the EB medium beginning at day 2 (lower row) at the same concentration used with the iPax3 ES cells, or not added at all (upper row). Cell contents were harvested at days 3, 4, and 5, and stained with Flk-1 and PDGFαR antibodies. Fluorescence intensity for Flk-1 (APC-conjugated) is indicated on the y axis and PDGFαR (PE-conjugated) on the x axis. (b)Morphology of monolayers resulting from the three non-paraxial mesoderm sorted cell fractions: PDGFαR–Flk-1–, PDGFαR–Flk-1+, and PDGFαR+Flk-1+ from uninduced (upper row) as well as induced (lower row) iPax3 EBs cells. (c) Real time RT-PCR expression analysis for MyoD and Myogenin in monolayer outgrowths of the four cell fractions: PDGFαR–Flk-1– (DN), PDGFαR–Flk-1+ (Flk-1), PDGFαR+Flk-1– (PDGFαR), and PDGFαR+Flk-1+ (DP), which were sorted from uninduced and induced day five iPax3 EBs. Transcripts are normalized to GAPDH. Error bars indicate standard errors from four independent experiments. (d) Table representing the percentage of cells expressing Myf5, MyoD, and MHC in the monolayers obtained from Pax3-induced unsorted and sorted cell fractions. Data are mean ± SE. For each cell fraction, five representative pictures (200X) were analyzed and the percentage of positive cells was calculated among total cells (> 300 cells).

a

MarkerCulturecondition

Pax3(%)

Myf5(%)

MyoD(%)

Myogenin(%)

MHC(%)

Proliferation 96.6 ±1.5

94.1 ±2.7

79.8 ±7.8

27.2 ± 1.2 35.4 ±5.9

Differentiation 13.1 ±0.4

27.2 ±1.2

24.3 ±1.3

72.2 ± 3.1 78.4 ±2.2

b

bFGF + dox EBM + doxc

Myf5

MyoD

MHC

0.00E+00

5.00E+06

1.00E+07

1.50E+07

1 2 3 4 5

Passage

Cou

nt

DoxbFGF+ dox No doxbFGF no dox

Supplementary Figure 4

d

0

0.005

0.01

0.015

0.02

0.025

0.03

00.00050.001

0.0015

0.0020.00250.003

0.0035

EBM+dox EBM no dox MDM+dox MDM no dox

Myf

5

00.0010.0020.0030.0040.0050.0060.0070.008

0

0.002

0.004

0.006

0.008

0.01

0.012

EBM+dox EBM no dox MDM+dox MDM no dox

EBM+dox EBM no dox MDM+dox MDM no dox EBM+dox EBM no dox MDM+dox MDM no dox

Des

min

Myo

geni

n

Moy

D

Supplementary Figure 4. Proliferation and differentiation of ES-derived myogenic progenitors in vitro. (a) Table representing the percentage of cells expressing Pax3,Myf5,MyoD, Myogenin, and MHC, in monolayers derived from Pax3-induced EB-derived PDGFαR+Flk-1– cells in the presence of proliferation and differentiation medium. Data are mean ± SE. For each cell fraction, five representative pictures (200X) were analyzed and the percentage of positive cells was calculated among total cells (> 300 cells). (b–c) Effect of bFGF in the expansion of myogenic progenitors obtained from Pax3-induced EB-derived PDGFαR+Flk-1– cells: (b) Growth curve of day 5 EB-derived PDGFαR+Flk-1– cultured in the presence of EB medium ± dox ± bFGF. (c) Immunohistochemistry analyses of these expanded cells (EBM+ dox ± bFGF) for Myf5, MyoD, and MHC. Cells are co-stained with DAPI (blue). Scale bar is 100 μm. (d) Relative levels of gene expression in differentiation-induced monolayers using the following growth conditions: EB medium + dox (no differentiation control), EB medium – dox, muscle differentiation medium (MDM) + dox, MDM – dox. Transcripts are normalized to GAPDH.

Supplementary Figure 4, cont.:

a

Supplementary Figure 5

b

Forc

e (g

)

31 32 33 34 350

5

1015

20

25

Time (s)

Control cells (PDGFαR+Flk-1+)PBS

40

20

035 36 37 38 39 40

Time (s)

No CTX

23 24 25 26 27 28 29

60

30

0

Time (s)

CTX

PBSTarget cells (PDGFαR+Flk-1–)

Forc

e (g

)

Forc

e (g

)

0

20

40

60

1

c

No CTX CTX

PBS Cells PBS CellsAbso

lute

For

ce-F

0(g

ram

)

**

***

PBS Cells

d

No CTX CTX

PBS Cells0

20

40

60

80

Spec

ific

Forc

e-sF

0 (k

N/m

2 )

*

*

h

010203040506070

0-6 6-12 12-18 18-24 24-30 >30

Myofiber CSA (X100µm)

010203040506070

0-6 6-12 12-18 18-24 24-30 >30

Myofiber CSA (X100µm)

No CTX CTXPBSCells

***

***

***

*******

***

***

Myo

fiber

CSA

dis

tribu

tion

(%)

e

0

30

60

90

120

Wei

ght (

mg)

No CTX CTX

PBS Cells PBS Cells

f

CS

A (m

m2 )

0

2

4

6

8

No CTX CTX

PBS Cells PBS Cells Fatig

ue in

dex

(s to

30%

Fo)

0

2

4

6g

No CTX CTX

PBS Cells PBS Cells

Supplementary Figure 5, cont.:

Supplementary Figure 5. Intramuscular transplantation of ES-derived myogenic progenitors. (a) Left panel, top row: Dystrophin expression (in red) in wild-type mice (positive control) as well as in PBS-treated mdx mice (negative control); Second and third rows: Teratoma formation in mdx mice transplanted with PDGFαR–Flk-1+ and PDGFαR–Flk-1– cell fractions. H&E staining and merge for DAPI, GFP and dystrophin is shown; Right panel, Top row: Transplantation of GFP – Pax3-induced EB-derived PDGFαR+Flk-1– cells generates GFP – Dystrophin+ myofibers (total of five mice). Lower row: Three consecutive serial sections (100µm apart) of a field containing Dystrophin+ myofibers in a mdx mouse treated with Pax3-induced PDGFαR+Flk-1– cells to demonstrate contiguous Dystrophin–positivity across a long section of muscle fiber. Scale bar is 100 μm. (b) Representative example of force tracing in TA muscles from mdx mice treated with control cells (PDGFαR+Flk-1+) or target cells (PDGFαR+Flk-1 –). Blue and red lines show force tracing from muscles that had received cell transplantation or PBS (control), respectively. (c-d) Effect of cell transplantation on absolute and specific force (sF0: F0 normalized to CSA). (e–f) Average CSA and weight of analyzed muscles, respectively. (g) Fatigue index – time for force to decline to 30% of maximal force during continuous stimulation of muscle at 150Hz. (h) Frequency histograms showing the distribution of myofiber CSA in PBS- and cell-treated in uninjured (left) and CTX-injured (right) mdx mice. 500 muscle fibers per experimental group were measured. (100 per mouse) * p <0.05, ** p <0.01, *** p <0.001

Aorta

Femoral artery

2nd ligation site (step 3)

Injection site

Blood flow toward targeted hind limb

Cells injected through catheter at aorta bifurcation site (step 2)Catheter

1st ligation site (step 1)

Supplementary Figure 6. Strategy for intra-arterial transplantation. The contra-lateral femoral artery was canulated retrogradely (left leg) toward the lumbar aorta. Cells were directly injected into the aorta at the level of iliac bifurcation followed by ligation of the contra-lateral artery to prevent bleeding. In this condition all blood flow is shifted into the targeted right limb. Despite ligation of the artery, due to collateral perfusion through superficial arteries, the left limb recovers and also shows some engraftment, although less than the targeted limb.

Supplementary Table 1. Summary of transplantation. Average frequency of dystrophin positive myofibers in mdx mice transplanted with PDGFαR+Flk-1– derived cells via intramuscular, intra-venous (tail vein) or intra-arterial route, in the presence or absence of CTX injury to the TA muscle, and among several control groups (mdx mice receiving no treatment, PBS, or negative cells: PDGFαR+Flk-1+). Measurements were also obtained from the gastrocnemius (GC). Data are Mean ± SE.

Groups Route Numberof cells

Number of mice

muscles CTX Dystrophin %

Untreated NA NA 5 R- TA - 2.6 ± 0.72 Untreated NA NA 5 R- TA + 3.1 ± 0.38

PBS Intramuscular NA 5 R- TA + 2.5 ± 0.42 PBS Tail vein NA 5 R- TA + 3.3 ± 0.51

Unsorted Intramuscular 106 5 R- TA + N.A (Tumor)

PDGFαR– Flk-1– Intramuscular 106 2 R- TA + N.A (Tumor)

PDGFαR–Flk-1+ Intramuscular 106 2 R- TA + N.A (Tumor)

PDGFαR+Flk-1+ Intramuscular 106 2 R-TA + 2.3 ± 0.62 PDGFαR+Flk-1– Intramuscular 106 5 R- TA - 10.8 ± 2.3 PDGFαR+Flk-1– Intramuscular 106 5 R- TA + 14.1 ± 2.4

PDGFαR+Flk-1–

Tail vein (I)

5 x 105 4 R- TA R- GC L- TA L- GC

+ - - -

12.6 ± 1.1 8.1 ± 0.85 7.7 ± 0.68 7.3 ±0.88

PDGFαR+Flk-1–

Tail vein (II)

5 x 105 5 R+L TA R+L GC

+ -

12.4 ± 0.59 9.8 ± 0.54

PDGFαR+Flk-1–

Intra-arterial 5 x 105 5 R- TA R- GC L- TA L- GC

+ - - -

15.9 ± 1.5 11.4 ± 1.2 7.1 ± 0.7 6.9 ± 0.7

1

SUPPLEMENTARY METHODS

Growth and differentiation of ES cells. ES cells were used in this study. ES cells were

maintained on irradiated mouse embryonic fibroblasts (MEFs) in knockout DMEM

(Invitrogen) supplemented with 1000 U/ml LIF (leukemia inhibitory factor; Chemicon),

15 % inactivated fetal bovine serum (Gemini), 0.1 mM non–essential amino acids

(Sigma), and 0.1 mM of beta–mercaptoethanol (Sigma). For differentiation cultures, ES

cells were trypsinized, resuspended in embryoid body differentiation (EB) medium,

IMDM supplemented with 15% FBS (Stem Cell Technologies), 4.5 mM

monothioglycerol (Sigma), 50µg/ml ascorbic acid (Sigma), and 200µg/ml iron saturated

transferrin (Sigma), and plated onto fresh gelatin coated T25 flasks for 45 min to allow

MEFs to adhere. Non–adhering cells (ES cells, depleted of MEFs) were then plated as

hanging drops in EB medium at a concentration of 100 cells per 10 μL drop in an

inverted bacterial Petri dish. After 48 hours in culture, EBs were collected and recultured

in 10 ml of EB medium in low adherence 10 cm Petri dishes on a slowly swirling table

rotator (set up inside of the tissue culture incubator). Slow rotation prevents the

attachment of the EBs to the culture dishes. At day 4, EBs were fed by exchanging half

of their spent medium for fresh EB medium. To induce Pax3 expression during EB

differentiation, doxycyclin (Sigma) was added to the cultures at 1µg/ml beginning at day

2.

Antibodies used for FACS analysis and sorting of EB derived cells. For PDGFαR, a

rat anti-mouse antibody was used (clone APA5; Pharmingen). For Flk-1, a biotinylated

anti-mouse antibody was used (clone Avas12a1; R&D Systems). For characterization of

2

the cells expanded in culture, we also used biotinylated anti-CD34 (clone RAM34;

eBioscience), biotinylated anti-Syndecan4 (clone KY/8.2; Pharmingen), rat anti-CD29

(clone KMI6; Pharmingen), rat anti-CXCR4 (Pharmingen), rat anti-C-met (clone

eBioclone4; eBioscience), rat anti-CD44 (eBioscience), mouse anti-M-cadherin (clone 5;

Pharmingen), and R-Phycoerythrin (PE)-conjugated anti-CD56 (clone MEM-188;

Biolegend) antibodies. For secondary staining, PE-conjugated goat anti-rat, PE-

conjugated goat anti-mouse Ig (Pharmingen) or Streptavidin-Allophycocyanin (APC)

(Pharmingen) was used.

Labeling of Pax3 inducible ES cells with EGFP. The FUGW lentiviral vector, which

expresses GFP from the ubiquitin promoter, has been used to generate GFP+ mice19. This

vector was cotransfected with packaging and coat protein constructs Δ8.91 and pVSVG,

respectively, into 293T cells using the FuGENE 6 transfection reagent (Roche). Virus-

containing supernatant was collected 48 hours after transfection, filtered through a 0.45

μm filter, concentrated, and used for infection of iPax3 ES cells. Supernatant was

removed the next day, and cells were passaged for 4 days, when the brightest 2% of GFP+

cells were FACS-deposited into single wells of a 96-well dish pre–plated with MEFs.

Wells with single ES cell colonies were harvested and expanded into clonal cell lines.

These clones were tested individually for GFP expression during an EB time course and

muscle differentiation to evaluate silencing. A clone that showed no silencing was

selected for in vivo studies. Conditioned medium from this clone was tested and found to

be negative for lateral transfer to 293T cells.

In vitro culture of sorted d5 EB cells. Following cell sorting, cell fractions were re–

aggregated for 2 days in low adherence swirling plates, and then transferred into 35mm

3

dishes to grow as monolayers, as described above, in the presence of EBD medium

containing doxycycline. Once the cells became confluent (approximately after 5 days),

the ability of these cells to undergo final maturation was evaluated by discontinuing

doxycycline and replacing the EBD medium with muscle differentiation medium, which

consisted of low glucose DMEM supplemented with 2% horse serum. After 1 week in

culture, cells were evaluated by immunofluorescence.

Real Time PCR analysis. Total RNA was isolated using Trizol (Invitrogen) as

recommended by the manufacturer. First strand cDNA was produced using Superscript II

reverse transcriptase (Invitrogen) with Oligo dT. 5 % of first strand reaction was used for

each ensuing PCR reaction. Real time PCR for muscle specific genes was performed

using probe sets from Applied Biosystems.

Transplantation studies. One day before transplantation, 75µl of cardiotoxin (10µM,

Sigma) was injected into the right tibialis anterior (TA) muscle of each mouse to induce

muscle injury. For Rotarod studies, both right and left TA muscles were injured. For

intramuscular transplantation, 24 hrs later, cells were injected (1 x 106 in 50µl of

phosphate-buffered saline) into right TA muscles of each group. As control, 50 µl of PBS

were injected in the left TA muscle of animals. For intra-venous transplantations, cells

were injected in the tail vein at 5 x 105 in 200ul PBS. For intra-arterial transplantation,

following anesthetization of animals with ketamine/xylazine, the contra-lateral femoral

artery (left leg) was canulated retrogradely toward the lumbar aorta and then cells were

directly injected into the aorta at the level of iliac bifurcation followed by ligation of the

contra-lateral artery to prevent bleeding (Supplementary Fig. 7). Cells (5 x 105 in 200ul

PBS) were delivered over a period of 10 minutes using an infusion pump (11 Pico Plus

4

Syringe Pump; Harvard Apparatus). For immuno-suppression, mdx mice received a daily

dose of 5mg/kg FK 506 (Tacrolimus; Sigma) intra-peritoneally (IP) from the day before

cell injection until the time of euthanasia (30 days or 90 days after transplantation). In

some experiments, mice were treated with dox, which was provided in their drinking

water at 1mg/ml for 1 week after transplantation.

Immunofluorescence staining of cultured cells and tissue sections. Primary antibodies

included GFP (Chemicon and Abcam), Pax3 (clone 274212; R&D Systems), Myf5,

Myogenin (clone F5D), MyoD (clone MoAb 5.8A) (all 3 from BD Biosciences), MHC

(Developmental Studies Hybridoma Bank), Desmin (Sigma), M–cadherin (Pharmingen),

CD44 (eBioscience), PDGFαR (Pharmingen), and dystrophin (Abcam or clone

MANDRA1 from Sigma). Cy2 and Cy3 (Jackson Immunoresearch Laboratories)

secondary antibodies were used. In some experiments, Alexa fluor 555 goat-anti-rabbit

(Molecular probes) was used as secondary staining for dystrophin (rabbit dystrophin from

Abcam) and Alexa fluor 488 goat-anti-chicken (Invitrogen) was used as secondary

staining for GFP (chicken dystrophin from Abcam).

Muscle preparation for mechanical studies. For the measurement of contractile

properties, mice were anaesthetized with sodium pentobarbitone (70 mg/kg I.P.) and

intact tibialis anterior (TA) muscles were dissected and placed in an experimental organ

bath filled with mammalian Ringer solution containing (mM): NaCl 120.5; NaHCO3

20.4; glucose 10; KCl 4.8; CaCl2 1.6; MgSO4 1.2; NaH2PO4 1.2; pyruvate 1.0, adjusted

to pH 7.4. The chamber was perfused continuously with 95% O2– 5% CO2 and

maintained at a temperature of 25 °C. The muscles were stimulated by an electric field

generated between two platinum electrodes placed longitudinally on either side of the

5

muscle (Square wave pulses 25 V, 0.2 ms in duration, 150 Hz). Muscles were adjusted to

the optimum length (Lo) for the development of isometric twitch force and a 5 min

recovery period was allowed between stimulations. Optimal muscle length (Lo) and

stimulation voltage (25 V) were determined from micromanipulation of muscle length

and a series of twitch contractions that produced maximum isometric twitch force. For

measuring fatigue time, muscles were stimulated for 1 minute and the time for force to

decline to 30% of Fo was measured. In brief, after determination of optimal muscle

length (Lo) and measurement of maximum isometric tetanic force, total muscle cross–

sectional area (CSA) was calculated by dividing muscle mass (mg) by the product of

muscle length (mm) and 1.06 mg/mm3, the density of mammalian skeletal muscle.

Specific force (sFo) was determined by normalizing maximum isometric tetanic force to

CSA. The CSA of myofibers was measured using ImageJ software (version 1.37;

National Institutes of Health [NIH]). 500 muscle fibers per experimental group were

measured (100 per mouse) 50. The data were plotted using sigmaplot software (version

10.0; 2006 systat software, Inc.).