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

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1,00

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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

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only DRG (n=102) DRG/mTERT (n=95)se

gmen

t num

ber

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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