Presentation on theme: "Lisbon, 29 th of July, 2009 Mesenchymal Stem Cell Transplantation in a Model of Peripheral Nervous System Demyelination."— Presentation transcript:
Lisbon, 29 th of July, 2009 Mesenchymal Stem Cell Transplantation in a Model of Peripheral Nervous System Demyelination
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).
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 – Lin neg /Sca-1 pos 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)
In vivo studies: I.v. transplantation of EGFP+ cells (MSC) (1-2x10 6 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 transplantation Functional 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 transplantation Mechanism 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 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 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 Hypothesis 2 MSC 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.
Acknowledgments Nerve 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