The aim of this work was the study of trophic properties of MSCs on sensitive neurons in culture. In particular we tested the MSCs’s capacity of myelination in order to use these cells for demyelinating disease therapies. MSCs are adult, multipotent and undifferentiated stem cells derived from bone marrow and isolated by plastic adherence. DRG from 15-day old Sprague-Dawley rat embryos were removed and cultured on a substrate of rat-tail collagen in AN2 medium supplemented with 5 ng/ml NGF. In a previous study our lab demonstrated that MSCs are able to rescue DRG dissociated neurons and to support their long-lasting survival in presence of NGF when co-cultured at direct contact. In this work we characterized cultured DRG neurons cellular death by means of cytofluorimetric, immunofluorescences and electron microscopy tecniques, demonstrating that neuronal cultures died for apoptosis and that MSCs protected neurons from this kind of death. We then investigate if the trophic role of MSCs was mediated by the release of soluble factors. We demonstrated that cocultures MSCs conditioned medium didn’t improve neuronal survival. We also investigated if MSC express constitutively on their surface some molecules able to improve neuronal survival by plating neurons on paraformaldehyde fixed MSCs (0,5%, 4%) and even in this case neurons died as the untreated control neurons. Therefore we concluded that the effect of MSCs on neuronal survival was mainly mediated by direct contact. We also observe in electron microscopy some point of contact between MSCs and neurons, and using a vital fluorescent cytoplasmatic stain we demonstrated the possibility of a passage of material between the two populations. By immunofluorescences we observed the presence only in cocultures of connexin 32, 36 and 42, which are proteins involved in neuronal gap junction formation. Connexin 32 was found in correspondence of neuronal and MSC membrane, suggesting that there were connection between neurons and MSC. We also demonstrated by electron microscopy the presence of axonal myelination only in neurons in cocultures. The myelin production was not due to the presence of satellite cells in our cultures, because we eliminated them with fluorodesoxyuridin that kills proliferating cells, as confirmed by immunofluorescences experiment with two markers of glial cells, GFAP and S100. Moreover the myelin production is neither due to transdifferentiation of MSCs, as demonstrated by immunofluorescences. The myelin presented in cocultures is few but it is compact and functional, presenting Node of Ranvier and morphological characteristic similar to myelin of neuronal and Schwann cells cocultures. In conclusion in this work we demonstrated that MSC can myelinate neuronal processes in culture, without transdifferentiating in Schwann cells. These results can be the base of more elaborate studies on human MSCs and on other types of neurons, like SNC neurons, to confirm the validity of MSC even on SNC, in order to identify a possible therapy for demyelinating disease with stem cells.

(2009). Cellule staminali mesenchimali (msc) nelle patologie demielinizzanti: studio in vitro della promozione della sopravvivenza di neuroni e della formazione della mielina da parte di msc. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).

Cellule staminali mesenchimali (msc) nelle patologie demielinizzanti: studio in vitro della promozione della sopravvivenza di neuroni e della formazione della mielina da parte di msc

RAVASI, MADDALENA
2009-12-17

Abstract

The aim of this work was the study of trophic properties of MSCs on sensitive neurons in culture. In particular we tested the MSCs’s capacity of myelination in order to use these cells for demyelinating disease therapies. MSCs are adult, multipotent and undifferentiated stem cells derived from bone marrow and isolated by plastic adherence. DRG from 15-day old Sprague-Dawley rat embryos were removed and cultured on a substrate of rat-tail collagen in AN2 medium supplemented with 5 ng/ml NGF. In a previous study our lab demonstrated that MSCs are able to rescue DRG dissociated neurons and to support their long-lasting survival in presence of NGF when co-cultured at direct contact. In this work we characterized cultured DRG neurons cellular death by means of cytofluorimetric, immunofluorescences and electron microscopy tecniques, demonstrating that neuronal cultures died for apoptosis and that MSCs protected neurons from this kind of death. We then investigate if the trophic role of MSCs was mediated by the release of soluble factors. We demonstrated that cocultures MSCs conditioned medium didn’t improve neuronal survival. We also investigated if MSC express constitutively on their surface some molecules able to improve neuronal survival by plating neurons on paraformaldehyde fixed MSCs (0,5%, 4%) and even in this case neurons died as the untreated control neurons. Therefore we concluded that the effect of MSCs on neuronal survival was mainly mediated by direct contact. We also observe in electron microscopy some point of contact between MSCs and neurons, and using a vital fluorescent cytoplasmatic stain we demonstrated the possibility of a passage of material between the two populations. By immunofluorescences we observed the presence only in cocultures of connexin 32, 36 and 42, which are proteins involved in neuronal gap junction formation. Connexin 32 was found in correspondence of neuronal and MSC membrane, suggesting that there were connection between neurons and MSC. We also demonstrated by electron microscopy the presence of axonal myelination only in neurons in cocultures. The myelin production was not due to the presence of satellite cells in our cultures, because we eliminated them with fluorodesoxyuridin that kills proliferating cells, as confirmed by immunofluorescences experiment with two markers of glial cells, GFAP and S100. Moreover the myelin production is neither due to transdifferentiation of MSCs, as demonstrated by immunofluorescences. The myelin presented in cocultures is few but it is compact and functional, presenting Node of Ranvier and morphological characteristic similar to myelin of neuronal and Schwann cells cocultures. In conclusion in this work we demonstrated that MSC can myelinate neuronal processes in culture, without transdifferentiating in Schwann cells. These results can be the base of more elaborate studies on human MSCs and on other types of neurons, like SNC neurons, to confirm the validity of MSC even on SNC, in order to identify a possible therapy for demyelinating disease with stem cells.
TREDICI, GIOVANNI
DRG neurons, myelin, MSC, connexins, apoptosis
BIO/16 - ANATOMIA UMANA
Italian
NEUROSCIENZE - 17R
22
2008/2009
(2009). Cellule staminali mesenchimali (msc) nelle patologie demielinizzanti: studio in vitro della promozione della sopravvivenza di neuroni e della formazione della mielina da parte di msc. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2009).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/7472
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