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Mesenchymal Stem Cells (MSCs)
1. How do MSCs contribute to repair in damaged 8ssues?
2. Are MSCs are “immuno-‐priviledged”? 2
Few MSCs (0.1-‐1%) remain at target site
Definite clinical benefit when MSCS are included in repair strategies
Can differen8ate into mature cells in vitro, but liKle evidence they differen8ate into mature cells in vivo
Immune Modula5on by MSCs
• Previous work showed that Toll-‐like receptors (TLRs), which recognize danger signals on immune cells, also exist on MSCs (Tomchuck+ 2008 Stem Cells)
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• Specific stimulation of TLR4 and TLR3 on MSCs led to secretion of immune-activating or immune-suppressing factors
• Also had effects on migration and invasion
Hypothesis
1. That MSCs exist on a MSC1 MSC2 spectrum
2. That these changes in phenotypes are controlled by TLR pathways
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hMSCs MSC2 MSC1
TLR-‐priming Protocol
LPS (10ng/mL) and poly(I:C) (1 ug/mL) were used as agonists for TLR4 an TLR3, respec8vely
(LPS MSC1; poly(I:C) MSC2)
1. hMSCs were grown to 60-‐70% confluency
2. TLR agonists were added to the medium for 1 hr or 24hrs -‐They believe that low levels for only 1hr mimics the
gradient of danger signals seen in vivo
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Outline of Experiments
Immune Modula8ng Proper8es
ECM Deposi8on
Differen8a8onMigra8on
Secre8on of Immune Modulators
Cytokines PGE2/IDO
TGFβ expression SMAD
Allogenic Co-‐culture of hMSCs and PBMCs
Understand Mechanism
Cytokine/Chemokine Secre5on Experiment
Methods: 1) Aeer priming and an addi8onal 48 hr of culture,
condi8oned medium was analyzed with Bio-‐Plex Cytokine Assays
2) IDO measured aeer 6hrs 3) Also transfected hMSCs with dominant nega8ve
plasmids for each TLR-‐receptor and repeated their cytokine assay
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TLR3 (MSC2) s8mula8on leads to Secre8on of Immune-‐Suppressing Factors
Figure 1: (A) secretion of immune-suppressing factors; (B) secretion following TLR3 or TLR4 inactivation by dominant-negative plasmid transfection
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TLR3 (MSC2) s8mula8on leads to Produc8on of Immune-‐Suppressing Factors
Figure 8
Cytokine Secre5on Results
• Immunosuppressive factors are elevated following TLR3 (MSC2) s8mula8on but mostly unchanged by TLR4 (MSC1) ac8va8on
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Migra5on Experiment
Methods 1) hMSCs were incubated with LPS, poly(I:C), CCL5
(150 ng/mL), or TNFα (1 ng/mL) for either 1 or 24 hr prior to loading onto Matrigel-‐coated inserts
2) Growth medium (16.5% serum) added to lower chamber
3) Migrated cells counted aeer overnight incuba8on
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Short-‐term TLR S8mula8on Enhances MSC migra8on
Figure 2: No differences in migration between MSC1 and MSC2
Migra5on/Invasion Results
– Short-‐term s8mula8on of TLRs promoted migra8on, and 24hr incuba8on inhibited migra8on • No differences between MSC1 and MSC2
– Short-‐term incuba8on with MSC1 or MSC2 cytokines (CCL5 and TNFa) inhibited migra8on, whereas 24hr incuba8on enhanced migra8on
– Complex pathways control migra8on
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MSC Differen5a5on Experiment
Methods: hMSCs were induced to differen8ate to chondrogenic, adipogenic, and osteogenic lineages for four weeks in the presence of TLR3 and TLR4 ligands
-‐Stained with Safranin’O, Oil Red O, or Alizarin Red
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Figure 3: Oil Red O and Alizarin Red staining Note: did not show chondrogenesis results because they were not affected
Differen5a5on Results
Neg. Cntrl
MSC1
MSC2
Differen5a5on Results
– TLR3 ac8va8on (MSC2) inhibits fat and bone differen8a8on
– TLR4 ac8va8on (MSC1) inhibits fat differen8a8on, but promotes bone differen8a8on
– Chondrogenesis was unaffected
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ECM Deposi5on Experiment
To evaluate the effects of TLR priming on a classical role of MSCs
Methods: MSCs primed for 1hr then incubated for an addi8onal 24hrs, then stained using an8bodies for collagen I and II and fibronec8n
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MSC1 MSC2
Figure 4: MSC1 produce more collagen whereas MSC2 produce more fibronectin (quantitation using ImageJ confirmed significant differences)
Understanding the Mechanism
• The next few experiments are more about trying to understand the mechanisms involved
• They looked at various signal pathways/protein expression:
1) TGFβ 2) SMAD 3) JAGGED
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TGFβ Secre5on
• Transforming Growth Factor β (TGFβ), a known immune-‐suppressing factor, also mediates collagen deposi8on
• hMSCs were cultured for an addi8onal 48 hr aeer TLR priming and then condi8oned media analyzed for TGFβ 1, 2 & 3 were detected by luminex immunoassay
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Figure 5: MSC2 secrete lower levels of TGFβ1, 2 and 3; no change in MSC1
TGFβ Secre5on Results
– TGFβ (1 and 3) expression diminished in MSC2 compared with MSC1 and unprimed hMSCs • Agrees with previous data that MSC1 deposited twice as much collagen as MSC2
• But contradicts the expecta8on that MSC2 should secrete more TGFβ
– They reason that TGFβ plays a smaller role in immune-‐modula8on than for immune cells
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Different effects of TLR s5mula5on on SMAD3 and SMAD7
• SMAD 3 is phosphorylated in response to TGFβ signaling
• SMAD 7 is involved in the nega8ve feedback of TGFβ signaling (antagonis8c)
Results: – SMAD3 is ac8vated in TLR4-‐primed but not TLR3-‐primed hMSCs
– SMAD7 expression is induced aeer TLR3 but not TLR4 s8mula8on of hMSCs
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MSC1: Higher SMAD3 More TGFβ higher collagen deposi5on MSC1: Lower SMAD7 More TGFβ higher collagen deposi5on MSC2: Higher SMAD7 Less TGFβ less collagen deposi5on MSC2: Lower SMAD3 Less TGFβ less collagen deposi5on
Allogenic co-‐culture of hMSCs and PBMCS
Methods: 1) T-‐lymphocytes among PBMCs, in the presence or
absence of isolated TLR-‐primed or unprimed MSCs, were resuspended and ac8vated with 1 mg of CD3/CD28 an8body beads (ac8vates T-‐cells)
2) Aeer 72 hr, the cells were stained with CD8-‐ or CD4-‐an8body (T-‐cell markers), and CFSE-‐label dilu8on of the CD8+ cells was assessed by flow cytometry analysis.
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T-‐Cell Ac8va8on
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Neg Cntrl
Figure 9A: MSCs (unprimed) are immuno-suppressive
T-‐Cell Ac8va8on
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Figure 9: Priming of MSCs into MSC1 reverses the reduction in T-cell activation; but MSC2 still suppress
Immune Suppression Results
– Unprimed MSCs suppress T cell ac8va8on
– MSC1 (TLR4) increase T cell ac8va8on – MSC2 (TLR3) suppress T cell ac8va8on
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hMSCs MSC1 MSC2 TLR3-‐priming TLR4-‐priming
Pro-‐Inflammatory
Immuno-‐suppressive
Immuno-‐suppressive
Summary of Key Findings
MSC2 (TLR3 priming) MSC1 (TLR4 priming) • Enhanced fibronec8n deposi8on • Secre8on of immune suppressive factors (IP-‐10, RANTES, IDO, PGE2) • Inhibits fat and bone differen8a8on • Maintained suppression of T-‐Cell ac8va8on
• Enhanced collagen deposi8on • Secre8on of pro-‐inflammatory mediators (IL6, IL8) • Promotes bone differen8a8on, but inhibits fat differen8a8on • Reversal of established MSC suppression of T-‐cell ac8va8on
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Conclusions
• Similar to monocytes, dendri8c cells, and other immune cells, MSCs are immunosuppressive un8l a pro-‐inflammatory role is required to promote 8ssue repair
• MSCs contribute to repair by immune modula8on proper8es rather than direct differen8a8on into new 8ssue
• TLR4-‐priming of hMSCs results in a pro-‐inflammatory phenotype (MSC1) – important for early injury responses
• TLR3-‐priming supports an immune suppressive phenotype (MSC2) – important for resolving the 8ssue injury
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Significance
• Immune-‐modula8ng biomaterials will affect more than just immune cells
• Why do pro-‐inflammatory s8muli promote osteogenic differen8a8on, and why do an8-‐inflammatory s8muli inhibit adipogenesis?
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