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)
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
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
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
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
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