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Therapeutic Capacity of Apoptotic Mononuclear Cell Secretome in Experimental Spinal Cord Injury Doctoral viva Thomas Haider, M.D. CHRISTIAN DOPPLER LABORATORY for the Diagnosis & Regeneration of Cardiac and Thoracic Diseases Medical

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Therapeutic Capacity of Apoptotic Mononuclear Cell Secretome in Experimental Spinal Cord Injury

Doctoral viva Thomas Haider, M.D.

CHRISTIAN DOPPLER LABORATORY for the Diagnosis & Regeneration of Cardiac and Thoracic Diseases Medical University of Vienna www.meduniwien.ac.at/applied-immunology Vienna, March 29, 2016

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Introduction

Stem Cell Therapy

J Clin Neurol 2016;12(1):14-20

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Introduction

J Clin Neurol 2016;12(1):14-20

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Introduction

  • Nat. Rev. Cardiol. 2010; 7: 204-215.
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The Dying Stem Cell Hypothesis

by Anker et al. up to 25% of all transplanted cells are in the state of apoptosis apoptotic cells induce transient immunosuppression

Myocardial Infarc.on

Necrosis

A3rac.on of immune cells Secre.on of pro-inflammatory cytokines Amplifica.on of inflamma.on

IL-1 IL-6 TNF-α

J Am Coll Cardiol. 2005 Nov 15;46(10):1799-802. J Clin Invest. 2001 Oct;108(7):957-62.

Introduction

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Introduction

Circ Res. 2008 Nov 21; 103(11): 1204–1219.

Stem Cell Engraftment and Transdifferentiation?

  • Low number of transplanted cells
  • Permanent engraftment not observable
  • Therapeutic effect in less than 72h
  • Secretome (CM) alone delivers comparable results
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Introduction

Stem Cells InternaIonal, Volume 2015, ArIcle ID 765846

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Introduction

European Heart Journal (2008) 29, 2851–2858 .

Comparability of BMCs and PBMCs Secretome

  • Secretome: Difference in 35 out of 174 analyzed factors
  • 25 secreted factors were higher in BMC-secretome
  • 10 secreted factors were higher in PBMC-secretome
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Suspensions of apopto.c PBMC control

Eur J Clin Invest. 2009 Jun;39(6):445-56. Irradiated cultured apopto.c peripheral blood mononuclear cells regenerate infarcted myocardium.

Centrifuga.on Lyophiliza.on

Introduction

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  • Attenuation of acute myocardial infarction

(Lichtenauer et al; Basic Res Cardiol. 2011)

Introduction

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  • Attenuation of acute myocardial infarction

(Lichtenauer et al; Basic Res Cardiol. 2011)

  • Immunosuppression in an experimental myocarditis model

(Hoetzencker et al; Eur Heart J. 2013)

Introduction

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  • Attenuation of acute myocardial infarction

(Lichtenauer et al; Basic Res Cardiol. 2011)

Introduction

  • Immunosuppression in an experimental myocarditis model

(Hoetzencker et al; Eur Heart J. 2013)

  • Inhibition of MVO and thrombocyte activation

(Hoetzencker et al; Bas Res Cardiol 2012)

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  • Wound healing in a murine and porcine wound model

(Mildner et al; PLoS One. 2013)

Introduction

  • Attenuation of acute myocardial infarction

(Lichtenauer et al; Basic Res Cardiol. 2011)

  • Immunosuppression in an experimental myocarditis model

(Hoetzencker et al; Eur Heart J. 2013)

  • Inhibition of MVO and thrombocyte activation

(Hoetzencker et al; Bas Res Cardiol 2012)

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  • Wound healing in a murine and porcine wound model

(Mildner et al; PLoS One. 2013)

Introduction

  • Attenuation of acute myocardial infarction

(Lichtenauer et al; Basic Res Cardiol. 2011)

  • Immunosuppression in an experimental myocarditis model

(Hoetzencker et al; Eur Heart J. 2013)

  • Inhibition of MVO and thrombocyte activation

(Hoetzencker et al; Bas Res Cardiol 2012)

  • Experimental stroke model

(Altmann et al; F1000Res. 2014 Jun 19 [revised 2014 Oct 28])

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Introduction

Altmann et al., F1000Res. 2014 Jun 19

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Introduction

Altmann et al., F1000Res. 2014 Jun 19

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Introduction

HLF γ-IR

  • - + +

0.0 0.5 1.0 1.5 ** HLF

[Relative expression]

γ-IR

  • +

20h

Beer et al. BMC Genomics 2014

Induction of Apoptosis

irradiated non-irradiated

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Introduction

pos C5a CD40 ligand G-CSF GM-CSF GRO-α CCL1 sICAM-1 IFN-γ pos IL-4 IL-5 IL-6 IL-8 IL-16 IL-17 IL-17E IL-23 IL-27 MIF MIP-1α MIP-1β PAI- 1 IL-2 MCP-1 IL-1ra IL-13 CXCL11 TNF-α sTREM-1 IL-1β IL-12 p70 CXCL10 IL-1α IL-10 RANTES pos IL-32α SDF-1 neg pos Activin A ADAMTS-1 Angi

  • geni

n Angiopoieti n-1 Angiopoieti n-2 Plas mino gen Amphir egulin Artem in pos TF CXCL16 CD26 EGF EG- VEGF CD10 5 Endo statin Endotheli n-1 FGF acidi c FGF basic FGF- 4 FGF- 7 GDN F GM- CSF HB- EGF HGF IGFBP

  • 1

IGFBP

  • 2

IGFBP- 3 IL-1β IL-8 TGF- β1 Lepti n MCP

  • 1

MM P-9 NRG 1-β1 Pentr axin 3 PD- ECG F PDG F-AA PDG F- AB/ BB Perse phin CXC L4 CXC L4 Prola ctin MIP-1 a MM P-8 Serpi n B5 PAI- 1 Serpi n F1 TIM P-1 TIM P-4 TSP- 1 TSP- 2 uPA Vaso hibin VEG F VEG F-C pos neg

2,5*10^6 IL-8 (pg/ml) 2305,8 ±136,4 GRO-alpha (pg/ml) 487,7 ±89,5 ENA-78 (pg/ml) 37857,5 ±12734,2 MCP-1 (pg/ml) 739,9 ±175,5 NAP-2 (µg/ml) 9,9 ±0,5 RANTES (pg/ml) 22251,2 ±3641,9 sICAM-1 (pg/ml) 2068,2 ±415,2 VEGF165 (pg/ml) 640,1 ±35,2 IL-16 (pg/ml) 1254,2 ±77,6 IL-1ra (pg/ml) 410,7 ±167,0 IL-10 (pg/ml) 7,1 ±0,5 IGF-I (pg/ml) 5,8 ±3,2 HGF (pg/ml) 72,9 ±19,1 FGF-2 (pg/ml) 534,2 ±11,6 TGF-beta (pg/ml) 87,3 ±20,4 MMP9 (pg/ml) 3612,3 ±597,7 MIF (pg/ml) 20147,5 ±1140,2 PAI-1 (pg/ml) 5060,6 ±3247,5 SDF-1 (pg/ml) 148,5 ±7,1

Lichtenauer et. Al Basic Res Cardiol. 2011 Nov;106(6):1283-97.

Compounds of the Secretome

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Introduction

Beer et al. BMC Genomics 2014

Compounds of the Secretome

  • Secreted Proteins
  • Oxidized Lipids
  • Extracellular vesicles
  • Microparticles
  • Exosomes
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Introduction

Spinal Cord Injury

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Introduction

Spinal Cord Injury

  • 50 per 1 million annually worldwide (200/year in AUT)
  • Average age of patients : 31 years
  • 50% experience total loss of motor function
  • 2 out of 3 – cervical spinal cord affected

J Neurosurg. 1991; 75(1): 15-26. J Neurotrauma. 2004; 21(10): 1355-70.

www.wikipedia.com www.schweizer-illustrierte.ch www.web.de

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Introduction

Spinal Cord Injury

The Spine Journal 2004; 4(4): 451-64.

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Introduction

Spinal Cord Injury – Treatment

  • Early surgical decompression
  • Corticosteroids (?) – Methylprednisolone (MP) regime
  • Rehabilitation

J Neurotrauma 2011; 28: 1371-1399

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Introduction

Spinal Cord Injury – Treatment

  • 30 years of SCI research -> 1 approved treatment option (MP)

aside from surgical therapy

  • Recommendation for the use of MP in SCI was revoked in 2013

by the AANS

J Neurotrauma 2011; 28: 1371-1399

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Introduction

The Spine Journal 2004; 4(4): 451-64.

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Introduction

  • Front. Cell. Neurosci. 2013; 4.
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Hypothesis

Compounds of secretome of apoptotic peripheral blood mononuclear cells exert therapeutic capacity

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Methods

Infinite Horizon Impactor

J Neurotrauma. 2003; 20(2): 179-93. J Neurotrauma. 1995; 12(1): 1-21.

Contusion Injury Model BBB-Score

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Methods

Venous Blood Withdrawal Cell Separa.on Irradia.on Centrifuga.on Incuba.on for 24h Lyophiliza.on Lyophilized MNC-secretomes Virus Elimina.on (GMP)

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Methods

Exp Neurol. 2015; 267: 230-242.

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  • RESULTS-
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Results

Exp Neurol. 2015; 267: 230-242.

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Results

Exp Neurol. 2015; 267: 230-242.

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Results

Exp Neurol. 2015; 267: 230-242.

n=4

A B

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Results

Exp Neurol. 2015; 267: 230-242.

n=4

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Results

Exp Neurol. 2015; 267: 230-242.

n=3

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Results

Exp Neurol. 2015; 267: 230-242.

n=3

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Results

Exp Neurol. 2015; 267: 230-242.

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Results

Exp Neurol. 2015; 267: 230-242.

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Previous Results

Basic Res Cardiol. 2011 Jun;106(4):645-55.

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Results

Exp Neurol. 2015; 267: 230-242.

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Results

Exp Neurol. 2015; 267: 230-242.

n=3

Beer et al. BMC Genomics 2014.

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Results

Exp Neurol. 2015; 267: 230-242.

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Summary

  • Secretome of apoptotic PBMCs previously attributed

with

  • Cytoprotection/Inhibition of apoptosis
  • Immunomodulaton
  • Inhibition of microvascular obstruction/

thrombocyte activation

  • Translatable to Spinal Cord Injury

Exp Neurol. 2015; 267: 230-242.

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Summary

  • TTND. 2016; 3:e1198.
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Summary

  • Secretome of apoptotic PBMCs lead to
  • Improvement of neurologic outcome
  • Attenuation of morphological damage
  • Improvement of vascularity
  • Recruitment of peripheral monocytes
  • > Multilayered therapy

Exp Neurol. 2015; 267: 230-242.

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Outlook

  • Crossing of the blood-brain barrier?
  • Alternative administration route/Combination with

novel apporaches

  • Mechanisms involved in monocyte recruitment?
  • Translation to bed-side
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Acknowledgments

Chris.an Doppler Laboratory

for Cardiac and Thoracic Diagnosis and Regenera.on

Hendrik Jan Ankersmit Patrick Altmann Andreas Miaerbauer Maahias Zimmermann Lucian Beer Mahdi Mohammad Kasiri

Ins.tute of Neurology/Neuropathology

Romana Höeberger Irene Leißer Eva Dassler Gerda Ricken Mirjam Lutz

Department of Dermatology

Michael Mildner Bahar Golabi Heinz Redl Jens Hartmann Markus Rossmann

Department of Transfusion Medicine

Beate Rüger Tanja Buchacher

Department of Pediatrics

Gert Lubec

LBI Experimental and Clinical Traumatology Center for Brain Research

Simon Hametner ChrisIan Gabriel

Red Cross Blood Transfusion Service Linz

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Addendum

Exp Neurol. 2015; 267: 230-242.

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Addendum

Exp Neurol. 2015; 267: 230-242.