Transplanted Donor Cells Rescue Contra-Lateral … · Only 25% of Nuclei are of Donor Origin! R L...
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Transplanted Donor Cells Rescue Contra-Lateral Chemo-ablated Muscle Bed MGMT- P140K Notexin + BCNU DONOR CELLS (i.m.) Notexin + BCNU SALINE (i.m.) R L O 6 BG (i.p.) Only 25% of Nuclei are of Donor Origin!
Transcript of Transplanted Donor Cells Rescue Contra-Lateral … · Only 25% of Nuclei are of Donor Origin! R L...
Transplanted Donor Cells Rescue Contra-LateralChemo-ablated Muscle Bed
MGMT-P140K
WT
Notexin + BCNUDONOR CELLS
(i.m.)
Notexin + BCNUSALINE
(i.m.)
R L
or
O6 B
G (i
.p.)
Only 25% of Nuclei are of Donor Origin!
R L
NotexinBCNUCELLS
Notexin BCNU
NOCELLS
14 Days Post-Transplantation(n = 8 /4)
R L R L
Muscle Injected with MGMT Donor Cells
Muscle Injected with Wild-Type Donor Cells
Transplanted Donor Cells Rescue Contra-LateralChemo-ablated Muscle Bed
Images have been removed for the printing purpose.
Right EDL Left EDL
Rescue of Contra-Lateral Chemo-ablated MuscleBed with Negligible Donor Cell Contribution
D14 Post-Transplantation
R L
NotexinBCNU
MGMT(P140K)CELLS
Notexin BCNU
NOCELLS
Imag
es ha
ve be
en re
moved
for th
e prin
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urpos
e.
Sca-1(
-) / C
D45(-)
Sca-1(
+) / C
D45(-)
Sca-1(-)/CD45(-)/CD34(+)• Expressed by Satellite cellprogenitor cells
• Intramuscular transplantationhas been shown to result insuccessful engraftment
Sca-1(+)/CD45(-)/CD34(+)• Expressed by MuscleDerived Stem Cells (MDSCs)
• Intramuscular injection hasbeen shown to result insuccessful engraftment
• Also expressed byMesoangioblasts and skeletalmuscle SP cells
Heterogeneity within CD34+ve Cells from RegeneratingSkeletal Muscle
Images have been removed for the printing purpose.
Sca-1(
-) / C
D45(-)
Sca-1(
+) / C
D45(-)
CD56(-)
/ ALP(+)
CD144(+
) / CD56
(+)
CD144(+
) / CD56
(-)
CD56(-)/ALP(+)/CD34(+)• Pericyte marker
• Intra-arterial injection hasbeen shown to result insuccessful engraftment indown stream muscles
CD144(+)/CD56(+)/CD34(+)• Myo-endothelial marker
• Successful engraftment uponintra-muscular injection
CD144(+)/CD56(-)/CD34(+)• Endothelial cell marker
Heterogeneity within CD34+ve Cells from RegeneratingSkeletal Muscle
Images have been removed for the printing purpose.
Sca-1(
-) / C
D45(-)
Sca-1(
+) / C
D45(-)
CD56(-)
/ ALP(+)
CD144(+
) / CD56
(+)
CD144(+
) / CD56
(-)
Pax7(+
)
CD133(+
)
Pax7(+)/CD34(+)• Satellite cell marker
• Expressed in both quiescentand proliferating Satellite cells
CD133 (+)/CD34(+)• Endothelial progenitor marker butshown to have myogenecity
• Systemic injection resulted insuccessful engraftment intodownstream muscles
Heterogeneity within CD34+ve Cells from RegeneratingSkeletal Muscle
Images have been removed for the printing purpose.
Sca-1(
-) / C
D45(-)
Sca-1(
+) / C
D45(-)
CD56(-)
/ ALP(+)
CD144(+
) / CD56
(+)
CD144(+
) / CD56
(-)
Pax7(+
)
CD133(+
)
CD11b(+)
CXCR4(+)
CD11b(+)/CD34(+)• Monocyte marker
• Shown to aid in phagocytosis
• Promotes muscle stem cellproliferation and muscleregeneration
CXCR4(+)/CD34(+)• Receptor for SDF-1 and expressedin endothelial progenitors
• SDF-1 is secreted from injuredskeletal muscles
• Has been shown to contribute tomyofiber formation
Heterogeneity within CD34+ve Cells from RegeneratingSkeletal Muscle
Images have been removed for the printing purpose.
Peault et al. (2007) Molecular Therapy
3. Adult (Somatic) Stem Cells - Plasticity?
1. Neural Stem Cells for Blood.2. Bone-Marrow Stem Cells for
Liver, Skin and Neurons.3. Blood Vessel Stem Cells for
Fat and Muscles.
Questions & Future Direction:
• What is the Identity and Location of the Responding Stem Cells ?1. Cells recruited from systemic circulation?
WT
NotexinNO
CELLS
Notexin BCNU
MGMT(P140K)CELLS
GFP+: Near ubiquitous expression of EGFPin cardiac and skeletal muscle
From ‘An improved method of parabiosis’ BunsterE. & Meyer R., Anat Rec 1933;57:339-343.
ParabiosisUNSW ACEC Approval 09/27A
Peripheral blood equilibrium isestablished 2-3 weeks post-surgery
Bunster & Meyer (1933) Anatomical Records
Brack (2007) Science
Parabiosis Model
Conboy (2005) Nature
Parabiosis Model
• Is the Pro-Regenerative Signal Systemic ?• Are the Responding Progenitors Recruited from Circulation ?
WT
NotexinBCNU
NOCELLS
Notexin BCNU
MGMT(P140K)CELLS
GFP+:Near-ubiquitousexpression of EGFP
Imag
es ha
ve be
en re
moved
for th
e prin
ting p
urpos
e.
Parabiosis Model
• Is the Pro-Regenerative Signal Systemic ?• Are the Responding Progenitors Recruited from Circulation ?
WT
NotexinBCNU
NOCELLS
Notexin BCNU
MGMT(P140K)CELLS
GFP+:Near-ubiquitousexpression of EGFP
Day -3 Day 0
Male Donor CellsMGMT(tg/tg)
- Notexin into TA & EDL- 3 Days of regeneration
Selectionof Donor Cells-CD34(+ve) cells
- 6 x 104 donor cells
Systemic Stem Cell Activation Signals
• What is the Identity and Location of the Responding Stem Cells ?1. Cells recruited from systemic circulation?2. Local stem cells activated upon receiving signal in systemic circulation?
WT
NotexinNO
CELLS
Notexin BCNU
MGMT(P140K)CELLS
GFP+: Near ubiquitous expression of EGFPin cardiac and skeletal muscle
Future Directions
• Transplantation of various donor sub-populations.→ Identify population(s) responsible for the systemic pro-regenerative signal
• Candidate chemokine/cytokine & receptor screening→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Quantitative RT-PCR→ FACS analyses
• Implantation of bio-degradable gels releasing recombinant protein(s)→ Alginate Gel, Atelocollagen, Fmoc-L-Phenylalanine-L-Phenylalanine (Fmoc-Phe-Phe)
• Screening of Candidate Cytokines & Chemokines using qRT-PCR- Honours Project, Animesh Singla→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Multiple time-point analyses (D1 - D2 - D3 - D4 - D5 - D7 - D14)→ Notexin injured vs Chemo-ablated→ alpha-SG null (dystrophic muscle under long-term de/regeneration cycle)
Work in Progress
• Screening of Candidate Cytokines & Chemokines using qRT-PCR- Honours Project, Animesh Singla→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Multiple time-point analyses (D1 - D2 - D3 - D4 - D5 - D7 - D14)
Work in Progress
Sca-1(
-) / C
D45(-)
Sca-1(
+) / C
D45(-)
CD56(-)
/ ALP(+)
CD144(+
) / CD56
(+)
CD144(+
) / CD56
(-)
Pax7(+
)
CD133(+
)
CD11b(+)
CXCR4(+)
Images have been removed for the printing purpose.
• Screening of Candidate Cytokines & Chemokines using qRT-PCR- Honours Project, Animesh Singla→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Multiple time-point analyses (D1 - D2 - D3 - D4 - D5 - D7 - D14)
Work in Progress
Mousegastrocnemius5dayspostfreeze‐injuryshowingVEGF(brown)staining.(Wagatsuma,MolCellBiochem2007)
MusclebiopsyfromDMDpaQent(Christovet.al.,MolBiolCell2007)
• Screening of Candidate Cytokines & Chemokines using qRT-PCR- Honours Project, Animesh Singla→ SDF-1, VEGF, TNF-α, IGF-1 etc.→ Multiple time-point analyses (D1 - D2 - D3 - D4 - D5 - D7 - D14)→ Notexin injured vs Chemo-ablated→ alpha-SG null (dystrophic muscle under long-term de/regeneration cycle)
Work in Progress
alpha‐SGnullDiaphragm
8‐weekold 8‐weekold 16‐weekold(Ducloset.al.,JCellBiol1998)
