Muscle Derived Stem Cells-Blinded

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Isolation of muscle-derived stem cells by a modified “preplate” technique  Slowly-adhering Cells (Muscle derived stem cells)

Skeletal Muscle biopsy

After 1 h Pre-plating technique

After 24 h

Rapidly-adhering cells myoblasts

J Cell Biol. 150:1085-99, 2000; J Cell Biol. 157:851-64, 2002; Nature Protocol, Protocol, 3, #9: 1501-1509, 2008.

 

Review of literature showing the utility of the preplate technique and differences among protocols. Cells isolated/ Acronyms 

Animal/ Strain 

Age/ Sex (if available 

Source of muscle tissue  

Enzymes used 

Substrate used 

Authority/Reference number  

Year 

Myogenic cell lines

Rats

Newborn

Thigh muscle primary cultures

Trypsin, 0.05% in Ca'+ Mg'+ free Earle's salt solution, pH 7.5

Uncoated

Richler and Yaffe/61

1970

Primary myoblasts

C57BL/6N, C3H/HeN,

2-5 days neonates

Forelimb and hindlimb muscles

Dispase grade II, 2.4 U/ml,

Collagen coated

Rando and Blau/60

1994

BALB/cAnN, C57BL/10, B6C3Fe, BALB/c/nu/nu mice

and collagenase class II, 1%; supplemented with CaCl2

MDSC

NormalC57Bl/6J

3-5 days

Hindlimb muscles

0.2% collagenase-type XI followed by dispase in HBSS and 0.1% trypsinEDTA in HBSS.

Collagen type I coated

Qu-Petersen et al./41

2002

Pluripotent stem cells (PPSCs)

Sprague –  –Dawley Dawley rats

6 months

Gastrocnemius and flexor  digitorum

Trypsin-EDTA buffer

Gelatin coated flasks

Romero-Ramos et al./ 68 

2002

MDSC

Normal Sprague-Da wley rats

3−6 weeks/female

Hind limbs (gastrocnemius) muscle

collagenase and trypsin XI, dispase

Collagen coated type 1

Hwang et al./

MDSC

Human

45-72 years/ Males and Females

Brachioradialis muscle

0.25% Trypsin –edetic-acid  –edetic-acid buffer 

Collagen type 1 coated

Primary muscle progenitor  cells

Fisher 344× Brown Norway rats

2 months/ male

Hind-limb muscles

1.25 mg/ml pronase

Uncoated

Machida et al./65

2004

Skeletal based precursors for cardiomyocytes (Spoc)

Normal C57Bl/6J

6- to 14-weeks/ male

Leg muscles

Collagenase type 2 (twice)

Uncoated

Winitsky et al./69

2005

Primary muscle cells

Wistar-rats

Newborn

Calf muscle

Following Qu-Petersen et al. 2002

Collagen-coated

Sun et al./67

2005

MDSC (Myospheres)

Mice

3-4 weeks

Hind-limb muscles

0.05%-0.25% trypsinEDTA in steps

Gelatin-coated

Sarig et al./70

2006

MDCs

Turkey (BUT-T9 strain)

7 days

Pectoralis muscle

0.12% Pronase E dissolved in 199 medium

0.1% gelatin-coated

Rouger et al./66

2007

Skeletal muscle CD34+/CD45-

GFP- transgenic mice

3-8 weeks

Whole muscle of thigh and lower leg

0.1% Collagenase type IA in DMEM-5-10% FBS

Uncoated

Tamaki et al./71

2008

MDSC

Normal C57Bl/6, and EGFP transgenic mice

2-8 weeks

Hindlimb muscles

0.2% Collagenase A followed by dispase

Collagen coated and Uncoated

64

 Alessandri et al./62

 Arsic et al./72

2004 2004

2008

Gharaibeh et al. Isolation of slowly adhering cells containing stem cells from murine skeletal muscle by the preplate

technique. Nature Protocols, Vol 3, #9: 1501 1509, 2008.  

Muscle-Derived Stem Cell (MDSC) Pax7 upregulation

Sca-1 + CD34 -/+ Bcl-2 +

all Myogenic Regulatory Factors



Quiescent Satellite Cell

m-cadherin -/+ c-Met + CD34 + (truncated isoform) Myf5 -/+ MyoD -/+ desmin -/+ Pax7 + VCAM-1 +

Myf5/MyoD involved

Activated Satellite Cell/  Proliferating Myogenic Precursor

References:  Beauchamp et al. (2000), Yoshida et al. (1998), Miller et al. (1999), Seale and Rudnicki (2000), Seale et al. (2000), Cornelison et al. (1997), Lee et al. (2000)

m-cadherin + c-Met + CD34 + (full length) Myf5 + MyoD + desmin + myogenin -/+

Terminal Differentiation Muscle fibers

MRF4 + Myogenin + CD34 Pax7 other MRFs +

 

osteoblasts  osteoblasts 

adipocytes   adipocytes

muscle cells (skeletal/cardiac) myofibroblasts

chondrocytes

hematopoietic/  endothelial cells

Postnatal Murine MuscleDerived Stem Cells (MDSCs) nerve cells/neurons/glial cells hepatocyte/urinary bladder cells Nat Cell Biol 5(7):640 – 6, 6, 2003; 2003; J Cell Biol 150:1085 – 99, 99, 2000; 2000; J Cell Biol 157:851 – 64, 64, 2002; 2002; Am J Pathol 161:895–907, 2002; 2002; Am J Pathol 164:1007–19, 2004; Stem Cells 2007;25:2302-2311

Am J Pathol 161:895  907, 9   07, 2002; 2002; Am J Pathol 164:1007  19, 1   9, 2004; Stem Cells 2007;25:2302 2311  

Muscle-derived stem cell applications: • Skeletal muscle repair after disease or injuries due to sports or military combat • Bone and cartilage repair • Cardiac repair

• Spinal cord and nerve repair • Other injuries

 

Muscle Anatomy and Histology

 

Normal Muscle Stained with H&E

200X

400X

 

Stem cell transplantation for muscle repair (DMD) • Normal muscle cells (RAC or satellite cells) and purified cells (SAC or MDSC) were isolated from skeletal muscle of normal mice • The same number of cells (300,000) was injected into the gastrocnemius of mdx  mdx mice mice (animal model for DMD) • The number of dystrophin-positive myofibers was monitored 10, 30, and 90 days after injection

 

Sat. cells

MDSC

J Cell Biol 157:851-64, 2002  

Can we improve bone healing with • Murine Leukemia VirusBMP-2 • MLV-BMP-4 • or Ad-BMP-2 

MDSCs? MDSCs seeded in Gelfoam 5

(5x10 cells/ 7-mm disk)

Implant into 6-mm calvarial defect 

3 and 6 weeks: X-ray and histological analysis

SCID or C57BL/6J mouse  

No Cells

+ Cells skull inside

+ Cells outside

3 wks

 

Can VEGF enhance the bone healing elicited by genetically engineered MDSCs expressing BMP4?

4 wks

 

Potential Mechanism behind the Improved transplantation capacity of MDSCs: Effect of stressful environment on fusion index

MDSCs’ fusion index not affected by stressful environment

Urish K et al. Mol. Biol. Cell. 2009

 

Potential Mechanism behind the Improved transplantation capacity of MDSCs: MDSCs’ Resistance to Stress Str ess  “Oxidative Stress Induced Apoptosis”  100

*

MDSCs

   s    l    l 75    e    C    c    i    t

Satellite Cells

*

50

   o    t    p    o    p 25    A      %

0 2 5 0

μM

100 μM [H2O2] in Culture Medium

5 0 0

μM

Control

 Apoptosis was measured using flow cytometry cytometry with annexin/PI staining 18hrs after H2O2 exposure

*

(p<0.05).

Oshima H et al. Mol Ther 12(6):1130-41, 2005  

Glutathione (GSH) level, a major antioxidant, may differ between MDSCs and satellite cells  Sat. Cells   n   o    i    t   a    l   u   p   o    P    f   o      %

Sat.cells

MDSCs

MDSCs have higher Levels of Reduced Glutathione Glutathion e after staining with w ith 5 µM MCB.

MDSCs + 50µM DEM

50µM of Diethyl maleate (DEM deplete GSH from MDSC and make it comparable to Satellite Cells.

MDSCs + DEM display a decrease regeneration capacity when compared to non-treated MDSCs

Urish K et al. Mol. Biol. Cell.

2009  

Differential engraftment ability exhibited by MDSCs

Sex-related differences in muscle regeneration capacity of male and female MDSCs

MDSC-M MDSC-F myogenic markers

Pax7

 —  

 —  

c-met

 —  

 —  

MyoD

 —  

 —  

desmin

 —   /+

 —   /+

CD34

>75%

>75%

Sca-1

>75%

>75%

CD45

 —  

 —  

C-kit

 —  

 —  

stem cell markers

hematopoietic lineage

 — :

less than 1%,  —   /+: 20-60% or variable, +: >60% or as indicated

 

Significant difference in terms of Regeneration Index:

female MDSCs were better than male MDSCs 

Blood and marrow stem cell recipients given maternal rather than paternal grafts

exhibit superior survival (Bone Marrow Marrow Transplant Transplant 28:375-80, 2001).   

Potential Mechanism behind Sex-Related Differences: MDSCs’ Resistance to Stress     2

8000   m    /   c   s    l    l 6000   e    C     y 4000    t    i   s   n   e    D 2000    l    l   e    C 0

*

* *

Male #1

  s    l    l   e    C   e    i    t   v   s   o    P   c    i    t   o    t   p   o   p    A

70 60

Control Hypoxia

Mal e #2

*

*

Fem ale #1

Fem ale #2

Control 1000 μM H2O2

50 40

*

30 20 10 0

Male #1

Female #1

Hypoxia: To test the possible effect of hypoxic conditions, 2 populations of female MDSCs and 2 populations of male MDSCs were cultured in 1% oxygen for 48 hours.  Apoptosis: To test the possible effect of oxidative stress, male and female MDSCs were grown for 24 hours under normal conditions and then were exposed to 1000 μM H2O2 for 18 hours. = p < 0.05

*

Deasy.. B et al. J. Cell Biology. Deasy Biology. Vol 177: 73-86, 2007  

Female MDSCs do a better job in skeletal muscle. Better self-renewal   Higher proliferation/delayed fusion  

Better resistance to stress   Less fibrosis   The high level of molecular and behavioral heterogeneity hete rogeneity exhibited by MDSC populations, and the sex-related differences could, at least lea st partially, partially, explain many of the conflicting results reported results reported in the literature on stem cell and progenitor cell biology. (How about age, background and etc)! Deasy.. B et al. J. Cell Biology. Deasy Biology. Vol 177: 73-86, 2007

 

Myocardial infarct repair: MDSCs vs. satellite cells Myocardial Infarction (MI) “Ligation of left anterior descending coronary artery”  

Cell Injection

Immediately after MI



3 x 105 cells / 30 µl Control: PBS only 



3 Injections border zone x 2 infarcted area x 1



10 µl / injection

SCID mice Echocardiography,, histology/LacZ staining, Echocardiography st aining, & immunohistochemistry

Cell Injection 1w

2w

MDSCs: n = 17 Satellite cells (myoblasts): n = 22 PBS: n = 16

6w

12w

Oshima H et al. Mol Ther 12(6):1130-41, 2005  

Cardiac engraftment: nLacZ & dystrophin

 

 

 

Do MDSCs become cardiac cells? Colocalization Colocalizati on of MDSCs with cardiac cell markers

Paracrine effects on host stem cell recruitment Angiogenesis Other?     C9%    H    M   - 8%

  s 7%    l    l   c   a   e    i   c    d     r 6%   +   a       Z   c 5%     c     a    h    t       L    i 4%     n   w    f    d 3%   o     e    %   i    l   z 2%   a   c   o 1%    l   o   c 0%

2 weeks  

Cardiac Contractility 

Control (PBS) Wall Motion: poor

MDSCs Wall motion: much better  better 

4 weeks

8 weeks

 

Myocardial infarct repair: MDSCs vs. satellite cells

MDSCs decreased the enlargement of  the left ventricular cavity.

MDSCs significantly improved systolic function.

Like MDSCs transplanted into skeletal muscle, MDSCs transplanted into cardiac muscle display an improved transplantation capacity

when compared with satellite cells (myoblasts).  

Human Muscle Derived Cells

 

Origin of muscle derived stem cells ce lls within the Blood Vessel Vessel wall Human Skeletal Muscle

FACS    4    3    D    C

CD146

Endothelial cells Myo-endothelial cells

Pericytes

Long Term Proliferation — Self-Renewing Self-Renewing — Multipotent Multipotent differe differentiationntiation Resistance to stress……Like murine MDSCs B 

skeletal muscle

bone

cartilage

 Zheng B et al. Nature Biotech. Biotech. 25, 9: 1025-1034, 2007  2007 

 

Crisan M et al. Cell Stem Cell. 2008 Sep 11;3(3):301-13

 

Improved cell Transpla Transplantation ntation with myogenic-endothelial cells  in skeletal muscle  Myogenic  Endothelial  Myogenic  -endothelial 

Human specific anti-ß Spectrin 250

Regeneration 

200

Index 

150

(Number of myofibers per 100,000 Injected cells)

100

 

(

significant difference, p< 0.05 )

50 0 CD 56+ /34/144- / 45-

Myogenic

CD 56- /34+ /144+ / 45-

CD 56+ /34+ /144+ / 45-

Endothelial 

Myogenic-  endothelial 

Like murine MDSCs MDSCs when compared compared with satellite cells (myoblasts) (myoblasts) after implantation implantation in skeletal muscle

 Zheng B et al. Nature Biotech. 25, 9: 1025-1034, 2007   

Improved cell Transpla Transplantation ntation with myogenic-endothelial cells  in cardiac muscle  (%)  Fractional Area Change 

Myo (n = 5, each group)

Endo Myo-endo  (

*

   2

8 6

† 

120 100 80 60 40 20 0

CD56+

0.05

CD56+ CD34+ CD144+

Myo-endo 

*

† VS. PBS, p < 0.05 A

2e-3 tR

1e-3

Normoxia Hypoxia

N

†  n u

* o m

3e-5 n

2e-5

A a e M

2

1e-5 0

Endo

P <

3e-3

† 

Myo

CD34+ CD144+

Gene Expression Analysis for Myo-Endo hMDCs

4

0

Regeneration Index

 VS. CD56+ and CD34+ /CD144+,

* VS. PBS, p < 0.05

10

140

PBS 

significant difference, p< 0.05 )

(n=5, each group)    )   m    +    (    1   /   m    3   s    D  e   r   u    C   t    0   c    0   u   r    1   t    S    X  

  s   r   e    b    i    f   o   y    M    f   o   r   e    b   m   u    N

Myo-endo

PBS

Vegf

Hgf  

ND ND

ND

b-Fgf 

Igf-I 

Like murine MDSCs when compared compared with satellite cells (myoblasts) (myoblasts) after implantation implantation in

cardiac muscle

Okada M et al. J. Am. Coll. Cardiol. In press. 2008

 

 Mesenchymal Stem Cells (MSC) 

 Multipotent Adult Progenitor Cells (MAPC)

Fat Derived  Stem Cells

Umbilical Cord  and Cord Blood Derived  Progenitor Cells 

 Muscle Derived Stem Cells (MDSC)

Adult Multi-lineage Stem Cells

…blood

vessels

 

Can blood vessel cells from human adipose tissue (vascular stroma) display a myogenic potential? Human abdominal white adipose tissue (WAT) recovered from cosmetic surgery 

1- Digest with collagenase for 45 min at 37ºC 2- Centrifuge and recover the “stroma vascular fraction”  3- isolation of blood vessel derived cells

 

Multi-lineage Mesodermal Potential of fat Perivascular Cells

Crisan M et al. Cell Stem Cell. 2008 Sep 11;3(3):301-13 11;3(3):301-13

 

 

Increasing the vascularity following vascularity of tissue may facilitate healing following injury by increasing the available available pool pool of MDSC’ MDSC’ss 

 Vascularity  V ascularity of a given given muscle can can be manipulated: manipulated: Gene therapy (eg. VEGF, sFLT-1); Exercise (training, immobilization); Neuromuscular electrical stimulation (NES)



NES commonly used modality: disuse atrophy , spasticity (CP, SC injury), Investigational Investigational uses for treatment of pain, dysphagia, scoliosis, denervation, strengthening in normal nor mal individuals



Has been shown to increase vascularity in treated muscle groups

Experimental group:  Analyzed for  for 

10 mice

Electrical stimulation (30min.) Muscle injury w/ of bilateral TA (4 sec. cardiotoxin 7mAmp,10 sec. rest) x 2 wks

TA harvested at 5 & 10 days

angiogenesis, regeneration & regeneration fibrosis

Control:  Analyzed for  for 

Muscle injury w/ cardiotoxin

8 mice

TA harvested at 5 & 10 days

regeneration & regeneration fibrosis

 

Effect of neuromuscular electrical stimulation on muscle healing Angiogenesis

Regeneration

CD31: Uninjured TA, E-stim vs. Ctl

% fibrosis prophylactic E-stim x 2 weeks (CTX injury)

700

500 400

Fibrosis

# Regenerate Myofibers/hpf Prophylactic E-stim x 2 weeks

* *

   l   s   e   c 300   +    1    3    D200    C   #

600

ctrl-- uninjured, no estim 5 Day s/p Estim-uninjured 10 day s /p estim-uninjured

100

*

*

  f 500   p   h   /   s400   r   e   b300   i   F   #200

ctl e-stim

*

   i   s 50   o   r    b    i 40    F   30    %

ctl

*

100

*

*

20 10 0 5d

Expermiental Group ctl vs. 5d: P<0.05 ctl vs. 10d: P<0.05 5d vs 10d: P=0.38

70 60

0

0

80

10d

Experimental Group 5d: p<0.05 10d: p<.05 5d vs. 10d: P=.36

Blue bars: control no e-stim.

5d

10d

Exerimental Group 5d: p<0.05 10d: p<0.001 5d vs. 10d: 0.98

Red bars: e-stim.

Neuromuscular electrical stimulation increases skeletal muscle capillarity, improve muscle

tx

regeneration and reduce muscle fibrosis after injury  

What is the relevance of these findings for peripheral nerve ? Surgical Technique“ Vein wrapping of scarred nerve”  Regenerated Nerve (4 wks) ♂R Femoral vein over ♀R Femoral nerve ♂R Femoral vein

♀R Femoral nerve

Xu J. et al. The Journal of Hand Surgery, 2000;(1)-93-103

Y Chromosome

Fluoromyelin  Axons=Neurofilament  Axons=Neu rofilament

Chromosome 12  

Can Human MDSC heal sciatic nerve defects (6-10 weeks) d

p A

proximal 

B

middle 

C

SC Ax

Ax

M

SC

   )   m    (   n 80   o   x   a 60    d   e    t   a 40   n    i    l   e   y   m 20    f   o   a 0   e   r    A

   2

D

E

   )   m    (   s   s   e   n    k   c    i    h    t   n    i    l   e   y    M    2

50

F

1.0

0.8

40

30

20

  o    i    t   a   r     g

0.6

0.4

10

0.2

0

0.0

M

Non-operated 

Non-operated 

hMDPCs  

No-operated 

hMDPCs 

hMDPCs 

 

Human MDCs-regenerated sciatic nerve is functional SFI=Sciatic Functional Index

 Bain GR et al. Plast. Plast. Reconstr Reconstr.. Sur Surg. g. 1989 #50

A

B

PBS

#43

PBS

0.5

   r    o    t    c    a    f    t    h    g    n    e    l    t    n    i    r    P

0.3

**

**  

0.2

§

*

§

*





§

0.0 0

hMDPCs #43

3

C 0.8    r    o    t    c    a    f    d    a    e    r    p    s    e    o    T

hMDPCs #50

7 Weeks after injury

#50

#43

9

13

PBS

0.6 * 

§



§

0.4



§



§

0.2 0.0 0

3

7

9

13

Weeks after injury

Untransplanted Control

#50

D 0

3

#43

P BS

7

9

13

0 -20

2-4 wks

6-8 wks

9-12 wks

12-14 wks

   I    F    S



§

-40 -60

 * §



§

-80 -100 Weeks after injury



§

 Lavasani M et al. In submission Oct. 2008

 

Stem cells will enable the development of new regenerative approaches for various tissues  tissues 

Limitations remain….   The lack of per permanent manent markers to isolate stem cells represent a limitation for this technology? Can resistance to stress be used as a distinguishing behavior to isolate stem cells?  The development of Liv Live e Cell Imaging technology to isolate stem cells based on behavior?

 

Bioinformatic Bioinforma tic Cell Cultur Culture e System Several regions selected in each well Multi-well plate     s     r     e      h     c     r     a     e     s     e      R

Bioreactor network server

Stage/ robot CCD Camera

Combinatorial Control, image processing and

informatics  

Proliferation Differentiation Migration  Apoptosis Necrosis Effect of  growth factors & cytokines

 

Cell death induced by serum starvation! Red Staining:TMR- Tetramethyl rhodamine methylcytofluorometric measurements of  mitochondrial membrane membrane potential in cells (label live cells )

Green staining: Pico Green stains DNA after cell death (Label dead cell nuclei)  

 

re-con on ng: ec s o un ax a mechanical strain on muscle-derived stem cells Flexercell® Tension Plus™ System  (FX-4000T™) 

Flexcell International www.flexcellint.com www.flexcellint.com  

MDSCs are plated in flexible bottom culture plates

 

Adult Muscle- Derived Stem Cell (MDSC) Isolation Tissue Specific Growth Factors Added (BMP, IGF VEGF, TGF, NGF…) 

MDSC expanded in culture

Muscle Biopsy

Multilineage Differentiation

Myogenic Neurogenic Osteogenic and

Chondrocytic

Hematopoetic

Adipogenic

 

How far are we from clinical applications based on this technology?

 

engineering, based onTissue muscle-derived stem cells, for treatment of  urologic dysfunction  A clinical trial for urinary incontinence was initiated in

Inject MDSCs

Isolate MDSCs

Toronto, Canada; Woman’s muscle stem cells were injected into their bladder sphincter in an effort to treat urinary incontinence!

 



Certified shipping container for sending the muscle biopsy to CMI at 4˚C and for returning the CMIAMDC product to the clinical site on dry ice

•Shipped on dry ice •Ready for injection •Excellent cell survival/ recovery

 

Lessons Learned 



 

Number of cells that can be isolated from human muscle biopsies How to send the muscle biopsy How can we grow the cells. How to shipped the cells (Excellent cell survival/ recovery) 



Bone Marrow Derived CD 34+ (FDA Trial , Approved) Muscle derived stem cells versus

myoblast (In progress…) 

 

Muscle Derived Stem Cells  





   

    



MDSCs are isolated by the preplate technique. MDSC marker profile shows that they probably gave rise to Satellite cells. They are multipotent- differentiating into multiple lineages and repairing tissues more effectively than myoblasts. MDSC can be transduced with genes (BMPs, VEGF, SFL-T, S FL-T, etc.) and can be incorporated into scaffolds. Their superior regenerative ability is probably due to higher resistance to oxidative stress. Testing this mechanism by experiments experiments with w ith GSH, DEM, NAC. Repair of myocardial infarction is probably due to paracrine effects Increasing vascularity of the muscle by VEGF, and e-stimulation has increased MDSCs regenerative ability. MDSCs vs. Myo/ Endo/ Myo-endothelial/ Pericyte fractions. Nerve repair and use of vein wrap. Clinical trials – Urinary incontinence. Live Cell Imaging laboratory a “behavior -morphology profile” to add to “marker” profiles. Improving engraftment by FlexCell “pre -conditioning” before implantation.  Future of muscle derived/ vessel derived stem cells.

 

The view from our building  

1818 - James Blundell performed the first successful transfusion of human blood

 

1901 - Karl Landsteiner, an Austrian physician, described the first three human Blood groups (A, B and O)

 

2010: Blood transfusion is a standard and safe procedure performed everywhere everywhere even in a moving ambulance

 

Examples of Technological advances: Number of functions

 

Higher efficiency, smaller size, etc.

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