Transcript Diapositivo 1

Mesenchymal Stem Cell Transplantation in a
Model of Peripheral Nervous System
Demyelination
Lisbon, 29th of July, 2009
Twitcher Mice
Model of Krabbe’s Disease
 Autosomal recessive
disorder;
 Deficiency of the lysosomal enzyme
β-Galactosylceramidase (GALC);
 Progressive
accumulation of Psychosine;
 Demyelination both in the PNS and CNS;
 Early death (~40
PND).
Current Therapies for Krabbe’s Disease
 Bone Marrow Transplantation is not sufficient to restore myelination;
 Umbilical Cord Blood transplantation in infantile patients still show abnormal PNS
involvement;
 Enzyme Replacement Therapy is unsuitable because the blood-brain barrier precludes
the entry of i.v. administered enzyme.
Aim of this study
Evaluate and characterize the therapeutic potential of adult
mesenchymal stem cells (MSC) transplantation for PNS remyelination
in leukodystrophies:
1. Characterization of MSC used in transplantation experiments;
2. Evaluation of the functional and neuropathological recovery of mouse models
after MSC transplantation;
3. Identification of the mechanism(s) through which transplanted cells induce
recovery.
Mesenchymal Stem Cells
 Minute fraction of the heterogeneous
nonhematopoietic cell population of
bone marrow;
 Easy to harvest, isolate and expand
from the adult bone marrow;
 Ability to participate/induce
remyelination.
Adapted from www.od.nih.gov/stemcell/figure3big.gif
Sources of Mesenchymal Stem Cells

Primary mouse MSC – isolated from the bone marrow of adult EGFP+ mice;
selection by plastic adherence.

Murine EGFP+ MSC cell line – Linneg/Sca-1pos MSC; immortalized by transfection
with telomerase reverse transcriptase (MSC-EGFP-mTERT cells).
MSC as a good cell source for participating/inducing remyelination
in Leukodystrophies
RT-PCR and FACS: cells express in vitro markers of glial/neural cell origin, as
well as neurotrophins
Express active GALC (the Twitcher´s deficient enzyme)
GALC
6,00
nmol/h/mg prot
5,00
4,00
3,00
2,00
1,00
0,00
TwS1
MSC
mTERT
Primary MSC transplantation
- Protocol In vivo studies:
I.v. transplantation of EGFP+ cells (MSC) (1-2x106 of MSC/mouse):
MSC i.v. delivery and
mock-transplanted at
≈ 24-25 PND.
Mice sacrificed at
humane endpoints
Functional
Analysis;
PCR analysis for EGFP
detection;
Nerve morphometry;
Galc activity;
Psychosine levels.
Primary MSC transplantation
Functional and Neuropathological analysis
No functional recovery (life span, body weigh or twitching severity) was found in
Twitcher mice transplanted with primary MSC cells;
Significant increase in the density of myelinated axons in the sciatic nerve;
mock
WT
MSC
myelinated axons/ mm2
35000
***
30000
*
***
25000
20000
15000
10000
5000
0
WT
mock
MSC
* p≤0,05; *** p≤0,001
Primary MSC transplantation
Mechanism Assessment
GALC activity was increased in sciatic nerve and spinal cord of MSC i.v. transplanted
twitchers;
Sciatic Nerve
**
0.8
**
0.7
0.6
0.5
***
0.4
0.3
0.2
0.1
0
WT
mock
***
1.2
Galc activity (nmol/h/mg protein)
Galc activity (nmol/h/mg protein)
0.9
Spinal Cord
MSC
***
1
0.8
0.6
**
0.4
0.2
0
WT
mock
MSC
** p≤0,01; *** p≤0,001
Increase in GALC activity was not abble to rescue the psychosine levels.
MSC Mechanism of Action
- Hypothesis I.v. transplantation of primary MSC promotes remyelination in the Twitcher PNS.
However...
Few EGFP+ cells (donor-derived) were found in demyelinated nerves;
Despite that GALC activity was increased in the nerves of recipient mice, no significant
decrease of psychosine levels was observed.
As such...
MSC probably promote recovery through a Paracrine Mechanism, unrelated to
GALC secretion, instead of through Transdifferentiation.
MSC Mechanism of Action
- Strategy -
To facilitate the study of the mechanism by which MSC induce remyelination in
Twitcher mice, MSC-EGFP-mTERT cell line was used
The effect of MSC-EGFP-mTERT on myelinating cells in vitro is currently being
assessed
MSC-EGFP-mTERT Promote Neurite Outgrowth
MSC-EGFP-mTERT cells were able to induce axonal growth in coculture, transwell system, with
sensory neurons
7
6
* p=1,8021E-05
5
segment number
4
3
2
1
0
only DRG (n=102)
Only DRG
DRG/mTERT (n=95)
700
* p=3,7366E-05
600
Longest Neurite (a.u.)

500
400
300
200
100
0
DRG/mTERT
only DRG (n=102)
DRG/mTERT (n=95)
MSC-EGFP-mTERT Promote Neurite Outgrowth
In vitro, MSC-EGFP-mTERT are unable to correct GALC levels in Twitcher Schwann cells
Hypothesis 1
MSC-EGFP-mTERT might exert their effect on axonal growth through a Neurotrophic Effect
This will be evaluated by inhibiting the biological activity of different neutrophins, in vitro,
using: antibodies against BDNF, NGF, NT3
AND
antibodies against neurotrophin receptors
Effect on axonal growth
Hypothesis 2
MSC might facilitate the axonal growth through an Immunomodulatory Action
Hence, the putative anti-inflammatory properties of MSC will be explored by measurement of
pro- and anti-inflammatory cytokines (Bio-Plex multiplex assay).
Conclusions and Perspectives
MSC induce remyelination in Twitcher mice through a Paracrine Mechanism
In vitro, MSC additionally promote Neurite Outgrowth
Determine the mechanism by which MSC induce axonal growth
To address MSC involvement in neurite outgrowth the Nerve Crush Model will be used
as an in vivo model nerve regeneration.
Acknowledgments
Nerve Regeneration Group,
IBMC, Porto:
Mónica Mendes Sousa
Catarina Miranda
INEB, Porto:
Perpétua Pinto-do-Ó
Filipa Franquinho
Dip. Medicina Interna,
Università di Roma Tor
Vergata, Rome, Italy:
Fernando Mar
Giancarlo Forte
Pedro Brites
Paolo di Nardo
Márcia Liz
Vera Sousa
Lysosome and Peroxisome
Biology Unit, IBMC, Porto:
Clara Sá Miranda
Institute of DNA Medicine, The Jikei
University School of Medicine, Tokyo,
Japan:
Shen Jin-Song