Muscle Derived Stem Cells-blinded

7/30/2019 Muscle Derived Stem Cells-blinded

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Skeletal Musclebiopsy

Pre-platingtechnique

Slowly-adhering Cell

(Muscle derived stem ce

After 1 h

Rapidly-adhering cells

myoblasts

Isolation of muscle-derived stem cells by amodified preplate technique

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

After 24 h


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Cells isolated/ Acronyms Animal/ Strain Age/ Sex (ifavailable

Source of muscle tissue Enzymes used Substrate used Authority/Referencenumber

Ye

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 19

Primary myoblasts C57BL/6N, C3H/HeN,

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

2-5 days neonates Forelimb and hindlimb muscles Dispase grade II, 2.4 U/ml,

and collagenase class II,1%; supplemented with

CaCl2

Collagen coated Rando and Blau/60 19

MDSC NormalC57Bl/6J 3-5 days Hindlimb muscles 0.2% collagenase-type XIfollowed by dispase in

HBSS and 0.1% trypsin-

EDTA in HBSS.

Collagen type Icoated

Qu-Petersen et al./41 20

Pluripotent stem cells(PPSCs)

SpragueDawley rats 6 months Gastrocnemius and flexordigitorum

Trypsin-EDTA buffer Gelatin coated flasks Romero-Ramos et al./68

20

MDSC Normal Sprague-Dawley rats 36 weeks/female Hind limbs (gastrocnemius)muscle

collagenase XI, dispaseand trypsin

Collagen type 1coated

Hwang et al./64 20

MDSC Human 45-72 years/ Malesand Females

Brachioradialis muscle 0.25% Trypsinedetic-acidbuffer

Collagen type 1coated

Alessandri et al./62 20

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 20

Skeletal based precursorsfor cardiomyocytes (Spoc)

Normal C57Bl/6J 6- to 14-weeks/male

Leg muscles Collagenase type 2 (twice) Uncoated Winitsky et al./69 20

Primary muscle cells Wistar-rats Newborn Calf muscle Following Qu-Petersen etal. 2002

Collagen-coated Sun et al./67 20

MDSC (Myospheres) Mice 3-4 weeks Hind-limb muscles 0.05%-0.25% trypsin-EDTA in steps

Gelatin-coated Sarig et al./70 20

MDCs Turkey (BUT-T9 strain) 7 days Pectoralis muscle 0.12% Pronase Edissolved in 199 medium

0.1% gelatin-coated Rouger et al./66 20

Skeletal muscleCD34+/CD45-

GFP- transgenic mice 3-8 weeks Whole muscle of thigh andlower leg

0.1% Collagenase type IAin DMEM-5-10% FBS

Uncoated Tamaki et al./71 20

MDSC Normal C57Bl/6, and EGFPtransgenic mice

2-8 weeks Hindlimb muscles 0.2% Collagenase Afollowed by dispase

Collagen coated andUncoated

Arsic et al./72 20

Review of literature showing the utility of the preplate technique and differences among protocols.

Gharaibeh et al. Isolation of slowly adhering cells containing stem cells from murine skeletal muscle by the pretechnique. Nature Protocols, Vol 3, #9: 1501-1509, 2008.


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chondrocy

muscle cells

(skeletal/cardiac)osteoblasts

Postnatal Murine Muscle-

Derived Stem Cells

(MDSCs)

adipocytes

hematopoietic/

endothelial cells

hepatocyte/urinary bladder cells

nerve cells/neurons/glial ce

myofibroblasts

Nat Cell Biol 5(7):6406, 2003; J Cell Biol 150:108599, 2000; J Cell Biol 157:85164, 2002;

Am J Pathol 161:895907, 2002; Am J Pathol 164:100719, 2004; Stem Cells 2007;25:2302-231


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Muscle-derived stem cell applications:

Skeletal muscle repair afterdisease or injuries due tosports or military combat

Bone and cartilage repair

Cardiac repair

Spinal cord and nerve repair

Other injuries


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Muscle Anatomy andHistology


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Normal muscle cells (RAC or satelcells) and purified cells (SAC orMDSC) were isolated from skeletalmuscle of normal mice

The same number of cells (300,000was injected into the gastrocnemiuof mdxmice (animal model for DM

The number of dystrophin-positive

myofibers was monitored 10, 30, an90 days after injection

Stem cell transplantation for musclerepair (DMD)


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J Cell Biol 157:851-64, 2

MDSC

Sat. cells


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MDSCs seeded in Gelfoam(5x105 cells/ 7-mm disk)

X-ray

andhistologic

analysis

Implant into6-mm calvarial defect

3 and 6weeks:

Can we improve bone healing withMDSCs?

MurineLeukemia Virus-BMP-2 MLV-BMP-4 or Ad-BMP-2

SCID or C57BL/6J mouse


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No Cells + Cells skull inside3 wks

+ Cells outsid4 wks


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Can VEGF enhancethe bone healing

elicited bygenetically

engineered MDSCsexpressing BMP4?


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

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

MDSCs fusion index notaffected by stressful

environment

P

t ti l M h i b hi d th I d t l t ti


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Oxidative Stress Induced Apoptosis

*

0

25

50

75

100

Control 100 M 2 5 0 M 5 0 0 M

%A

poptoticCells MDSCs

Satellite Cells

**

[H2O2] in Culture Medium

Apoptosis was measured using flow cytometry with annexin/PI staining18hrs after H2O2 exposure

(p


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Glutathione (GSH) level, a major antioxidant, may differ

between MDSCs and satellite cellsSat.cells MDSCs

%

ofPopulatio

n

MDSCs have higher Levels of ReducedGlutathione after staining with 5 M MCB.

MDSCs

+ 50M DEM

Sat. Cells

50M of Diethyl maleate (DEM deplete GSH froMDSC and make it comparable to Satellite Ce

MDSCs + DEM display adecrease regeneration capawhen compared to non-trea

MDSCs

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


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Significantdifference in terms

of RegenerationIndex:

female MDSCswere betterthan male

MDSCs

Blood and marrow stem cell recipients given maternal rather than paternal graftexhibit superior survival (Bone Marrow Transplant 28:375-80, 2001).


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Female MDSCs do a better job in skeletal


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Better self-renewal

Better resistance to stress

Female MDSCs do a better job in skeletalmuscle.

Less fibrosis

The high level of molecular and behavioral heterogeneityexhibited by MDSC populations, and the sex-related

differences could, at least partially, explain many of the

conflicting resultsreported in the literature on stem cell andprogenitor cell biology.(How about age, background and etc)!

Higher proliferation/delayed fusio

Deasy. B et al. J. Cell Biology. Vol 177: 73-86, 2


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Cardiac engraftment: nLacZ& dystrophin


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Myocardial infarct repair: MDSCs vs. satellite cells


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MDSCs

significantly

improved

systolic

function.

MDSCs

decreased the

enlargement of

the left

ventricularcavity.

Myocardial infarct repair: MDSCs vs. satellite cells

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


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Human Muscle Derived Cells


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Improved cell Transplantation with myogenic-endothelial cells


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Human specific anti- Spectrin

Endothelial

Myogenic-endothelial

Myogenic in skeletal muscle

Myogenic-

endothelial

250

200

150

0

100

50

Myogenic Endothelial

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

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

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

Regeneration

Index( significant difference,

p< 0.05 )

(Number of myofibersper 100,000 Injected cells)

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

skeletal muscle

Zheng B et al. Nature Biotech. 25, 9: 1025-1034, 200

Improved cell Transplantation with myogenic-endothelial cellsin cardiac muscle


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( significant difference, p< 0.05 )

(%) Fractional Area Change

(n = 5, each group)

Myo Endo Myo-endoPBS

0

2

4

6

8

10

(n=5, each group)

X100CD

31(+)

Structures/mm2

Myo Endo Myo-endo PBS

*

VS. PBS, p < 0.05

VS. PBS, p < 0.05*

0

20

40

60

80

100

120

140

CD56+ CD34+

CD144+

CD56+

CD34+

CD144+

Regeneration Index

Numbero

fMyofibers

VS. CD56+ and CD34+ /CD144+, P< 0.05

Gene Expression Analysis for Myo-Endo hMDCs

MeanAmountRNA

0

1e-5

2e-5

3e-5

1e-3

2e-3

3e-3 NormoxiaHypoxia

Vegf Hgf b-Fgf Igf-I

ND ND ND

*

*

Myo-endo

Okada M et al. J. Am. Coll. Cardiol. In press. 2

in cardiac muscle

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

cardiac muscle


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Muscle Derived Stem Cells (MDSC)

Multipotent Adult

Progenitor Cells(MAPC)

Adult Multi-lineage Stem Ce

Fat Derived

Stem Cells

MesenchymalStem Cells (MS

Umbilical Cordand Cord Blood DerivedProgenitor Cells

blood vessels


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Multi-lineage Mesodermal Potential of fat Perivascular Cells


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g

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

Increasing the vascularity of tissue may facilitate healing following


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10 mice Electrical stimulation (30min.)of bilateral TA (4 sec.

7mAmp,10 sec. rest) x 2 wks

Muscle injury w/

cardiotoxin

TA harvested at

5 & 10 days

Analyzed for

angiogenesis,

regeneration &

fibrosis

Experimental group:

Control:

8 miceMuscle injury w/

cardiotoxin

TA harvested at

5 & 10 days

Analyzed fo

regeneration

fibrosis

NES commonly used modality: disuse atrophy , spasticity (CP,SC injury), Investigational uses for treatment of pain, dysphagia,

scoliosis, denervation, strengthening in normal individuals

Has been shown to increase vascularity in treated muscle groups

g g g

injury by increasing the available pool of MDSCs

Vascularity of a given muscle can be manipulated: Gene therapy (eg. VEGF, sFLT-1);

Exercise (training, immobilization); Neuromuscular electrical stimulation (NES)

Eff t f l l t i l ti l ti l h li


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Neuromuscular electrical stimulation increases skeletal muscle capillarity, improve muscleregeneration and reduce muscle fibrosis after injury

Effect of neuromuscular electrical stimulation on muscle heali

CD31: Uninjured TA, E-stim vs. Ctl

0

100

200

300

400

500

Expermiental Group

ctl vs. 5d: P


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Surgical Technique Vein wrapping of scarred nerve

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

Fluoromyelin

Axons=Neurofilament

Regenerated Nerve (4 wks)

R Femoral vein over R Femoral nerve

Y Chromosome

Chromosome 12

g p p

R Femoral vein

R Femoral nerve

Can Human MDSC heal sciatic nerve


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pd

defects (6-10 weeks)

Ax

SC

M

m

Ax

SC

M

proximal

0.0

0.2

0.4

0.6

0.8

1.0

g-ratio

hMDPCsNo-operated

0

10

20

30

40

50

Myelinthickness(m2)

hMDPCsNon-operated

0

20

40

60

80

Areaofmyelinatedaxon(m2)

hMDPCsNon-operated

A B C

D E F

Human MDCs-regenerated sciatic nerve is functional


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0.0

0.2

0.4

0.6

0.8

0 3 7 9 13

Weeks after injury

Toe

spread

factor

#50 #43 PBS

* * **

0.0

0.2

0.3

0.5

0 3 7 9 13

Weeks after injury

Printlenght

factor

#50 #43 PBS

** *

**

*

**

-100

-80

-60

-40

-20

0

0 3 7 9 13

Weeks after injury

SF

I

#50 #43 PBS

*

***12-14 wks2-4 wks 9-12 wks6-8 wks

PBS

hMDPCs #43

hMDPCs #50

Untransplanted

Control

A B

C

D

gSFI=Sciatic Functional Index

Bain GR et al. Plast. Reconstr. Surg. 19

Lavasani M et al. In submission Oct. 200


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Stem cells will enable the development of new regenerativ


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Stem cells will enable the development of new regenerativ

approaches for various tissues

Can resistance to stress be used as a distinguishing

behavior to isolate stem cells?

The lack of permanent markers to isolate stem cells

represent a limitation for this technology?

Limitations remain.

The development of Live Cell Imaging technology toisolate stem cells based on behavior?

Bi i f ti C ll C lt S tBi i f ti C ll C lt S t


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Bioinformatic Cell Culture System

Stage/ robot

Multi-well plate

Several regionsselected in each well

Combinatorial Control,image processing andinformatics

CCDCamera

Bioreactornetworkserver

Researchers

Bioinformatic Cell Culture System

Stage/ robot

Multi-well plate

Several regionsselected in each well

Combinatorial Control,image processing andinformatics

CCDCamera

Bioreactornetworkserver

Researchers


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Proliferatio

Differentiat

Migration

Apoptosis

Necrosis

Effect of

growthfactors &

cytokines


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Red Staining:TMR- Tetramethyl rhodamine methylcytofluorometric measurements of

mitochondrial membrane potential in cells (label live cells )

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

Cell death induced by serum starvation!

re-con on ng: ec s o un ax amechani

cal strain on muscle-derived stem


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MDSCs are plated in flexibbottom culture plates

Flexercell Tension Plus System

(FX-4000T)

Flexcell International www.flexcellint.com

mechanical strain on muscle-derived stemcells

Adult Muscle- Derived Stem Cell(MDSC) Isolation
http://www.flexcellint.com/http://www.flexcellint.com/


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Myogenic

Osteogenicand

Hematopoetic Adipogenic

Chondrocytic

Neurogeni

Muscle Biopsy

Tissue Specific GrowthFactors Added (BMP, IGFVEGF, TGF, NGF)

MDSC expanded in culture

Multilineage Differentiation

How far are we from clinical applications


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pp

based on this technology?


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Isolate

MDSCs

Inject

MDSCs

Tissue engineering, based

on muscle-derived stem cells

for treatment ofurologic dysfunction

A clinical trial for urinary

incontinence was initiated in

Toronto, Canada; Womans

muscle stem cells were injecte

into their bladder sphincter in

an effort to treat urinaryincontinence!


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Certified shippingcontainer for sendingthe muscle biopsy to

CMI at 4C and forreturning the CMI-AMDC product to theclinical site on dry ice

Shipped on dry ice

Ready for injection

Excellent cell

survival/ recovery

Lessons Learned


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Lessons Learned Number of cells that can be

isolated from human

muscle biopsies

How to send the musclebiopsy

How can we grow the cells.

How to shipped the cells(Excellent cell survival/recovery)

Bone Marrow Derived CD 34+ (FDTrial , Approved)

Muscle derived stem cells versusmyoblast (In progress)

Muscle Derived Stem Cells


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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, etc.) and can be incorporatedinto scaffolds.

Their superior regenerative ability is probably due to higher resistance to oxidative stress.

Testing this mechanism by experiments with 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.


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The view from our buildi

1818 - James Blundell performed the first successfultransfusion of human blood


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


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g p ( )

2010: Blood transfusion is a standard and safe procedure

f d h i i b l


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performed everywhere even in a moving ambulance

Examples of Technological advances: Number of functions


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Higher efficiency, smaller size, etc.


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Documents

Muscle Derived Stem Cells-blinded