mesenchymal stem cell transplantation in a model of peripheral nervous system demyelination
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Lisbon, 29th of July, 2009
Mesenchymal Stem Cell Transplantation in a Model of Peripheral Nervous System
Demyelination
Twitcher MiceModel 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).
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.
Current Therapies for Krabbe’s Disease
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).
RT-PCR and FACS: cells express in vitro markers of glial/neural cell origin, as well as neurotrophins
MSC as a good cell source for participating/inducing remyelination in Leukodystrophies
Express active GALC (the Twitcher´s deficient enzyme)
GALC
0,00
1,00
2,00
3,00
4,00
5,00
6,00
TwS1 MSC mTERT
nmol
/h/m
g pr
ot
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
PCR analysis for EGFP detection;Nerve morphometry;Galc activity;Psychosine levels.Functional
Analysis;
Primary MSC transplantation- Protocol -
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;
WT mock
MSC
*** ***
*
Primary MSC transplantationFunctional and Neuropathological analysis
* p≤0,05; *** p≤0,001
GALC activity was increased in sciatic nerve and spinal cord of MSC i.v. transplanted twitchers;
Sciatic Nerve
***
****
Increase in GALC activity was not abble to rescue the psychosine levels.
**
***
Spinal Cord***
Primary MSC transplantationMechanism Assessment
** p≤0,01; *** p≤0,001
MSC Mechanism of Action- Hypothesis -
I.v. transplantation of primary MSC promotes remyelination in the Twitcher PNS.
Few EGFP+ cells (donor-derived) were found in demyelinated nerves;
However...
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
0
1
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7
only DRG (n=102) DRG/mTERT (n=95)se
gmen
t num
ber
0
100
200
300
400
500
600
700
only DRG (n=102) DRG/mTERT (n=95)
Long
est N
eurit
e (a
.u.)
DRG/mTERT
Only DRG
* p=1,8021E-05
* p=3,7366E-05
MSC-EGFP-mTERT Promote Neurite Outgrowth
In vitro, MSC-EGFP-mTERT are unable to correct GALC levels in Twitcher Schwann cells
Hypothesis 1MSC-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
Hypothesis 2MSC might facilitate the axonal growth through an Immunomodulatory Action
Effect on axonal growth
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.
AcknowledgmentsNerve Regeneration Group,
IBMC, Porto:
Mónica Mendes Sousa
Catarina Miranda
Márcia Liz
Filipa Franquinho
Fernando Mar
Pedro Brites
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
INEB, Porto:
Perpétua Pinto-do-Ó
Dip. Medicina Interna, Università di Roma Tor Vergata, Rome, Italy:
Giancarlo Forte
Paolo di Nardo
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