muscle derived stem cells-blinded
TRANSCRIPT
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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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