Recent findings indicate that stem cells could be a good new tool to reduce the secondary degeneration in spinal cord injury (1, 2). Amniotic fluid (AF), which contains differentiated and undifferentiated cells arising from all three germ layers, might be a novel, large and accessible reservoir of stem cells for therapeutic use (3, 4). The aim of this study was to use cells isolated from the third trimester amniotic fluid (as novelty in comparison to the cells obtained from amniocentesis) (see Abstract Bottai et al. SIF 2009) as therapeutic tool in a mouse model of spinal cord injury. By immunocytochemical and cytofluorimetric analysis three cultures were selected to be tested in in vivo studies. All these cultures showed a mesenchymal phenotype; the #3.5 and # 3.6 were directed to the muscle-neural lineage (the #3.6 also expressed CD117) and the #1.1 was directed to the perivascular lineage. The injury (contusion) was performed at T8 level by mean of the Infinite Horizon Device, that allows the generation of reproducible traumatic lesion to the cord. The animals were divided into four groups: 1) lesioned mice transplanted with PBS (control group), 2) lesioned mice transplanted with cells from the #1.1 culture, 3) lesioned mice transplanted with cells from the #3.5 culture and 4) lesioned mice transplanted with cells from the #3.6 culture. All the transplanted cells, injected intravenously one week after the lesion, were labelled using Quantum dot (Invitrogen). The hindlimb motor recovery was evaluated for the 35 days following the lesion according to the Basso Mouse Scale (6). From 14 days after transplantation, the cell cultures #3.6 and #3.5 induced a better recovery of motor function than PBS, whereas cell culture #1.1 failed. Spinal cord transversal sections were analyzed for the presence of Qdot labelled transplanted AF cells and for the expression of neural-glial markers. One month after i.v. transplantation several Qdot positive cells at the site of injury were found and some of these Qdot labelled cells were also positives for β-tubulin III and GFAP immunostaining. Furthermore, the histological analysis of the lesion site showed that animals transplanted with culture #3.6 had a better preserved ventral myelin, and the number of infiltrated inflammatory cells (macrophages and neutrophils) was also reduced in comparison to the control PBS treated animals. Transplanted cells were detected at the lesion site and 4 mm away in rostral position to the injured area. In a complementary set of experiments we transplanted AF cells into a spinal cord which was subjected to the hemisection at T8 level. Animals were divided into three groups: 1) lesioned mice injected with PBS (control group), 2) lesioned mice treated with intraspinal administration of AF cells (#3.6 culture) embedded in hydrogel and 3) lesioned mice treated with intraspinal administration of hydrogel alone. At one week after injury AF cells-hydrogel treated mice showed a better motor recovery compared to hydrogel and PBS treated mice, and the difference was very significant at 28 days. The hydrogel treated mice also presented a slight motor improvement than PBS group, but this increase was lower than that induced by AF cells-hydrogel treatment. Confocal analysis of transversal sections revealed the presence of Qdot positive cells surrounding the lesion site, indicating that AF cells migrated out of hydrogel and survived over one month after transplantation. These data imply that the administration of amniotic fluid derived cells with a muscle-neural phenotype can attenuate secondary degeneration following traumatic lesion to spinal cord by dropping the cellular component of neuroinflammatory reaction. References: 1. Reier PJ (2004) NeuroRx 1:424-451 2. Schultz SS (2005) Curr Drug Targets 6:63-73 3. Gosden CM (1983) British Medical Bullettin 39(4):348-354 4. Prusa AR et al. (2004) Am J Obstet Gynecol 191(1) 309-314 5. De Coppi P et al. (2007) Nature Biotecnology 25(1):100-105 6. Basso DM et al. (2006) J Neurotrauma 23:635

Third trimester human amniotic fluid cells with potential for therapy in neurodegenerative disorders / D. Cigognini, E. Nicora, E. Ripamonti, R. Adami, M. Menarini, A.M. Di Giulio, D. Bottai, A. Gorio. ((Intervento presentato al 34. convegno Congresso Nazionale della Società Italiana di Farmacologia tenutosi a Rimini nel 2009.

Third trimester human amniotic fluid cells with potential for therapy in neurodegenerative disorders

D. Cigognini
Primo
;
E. Nicora
Secondo
;
R. Adami;A.M. Di Giulio;D. Bottai
Penultimo
;
A. Gorio
Ultimo
2009

Abstract

Recent findings indicate that stem cells could be a good new tool to reduce the secondary degeneration in spinal cord injury (1, 2). Amniotic fluid (AF), which contains differentiated and undifferentiated cells arising from all three germ layers, might be a novel, large and accessible reservoir of stem cells for therapeutic use (3, 4). The aim of this study was to use cells isolated from the third trimester amniotic fluid (as novelty in comparison to the cells obtained from amniocentesis) (see Abstract Bottai et al. SIF 2009) as therapeutic tool in a mouse model of spinal cord injury. By immunocytochemical and cytofluorimetric analysis three cultures were selected to be tested in in vivo studies. All these cultures showed a mesenchymal phenotype; the #3.5 and # 3.6 were directed to the muscle-neural lineage (the #3.6 also expressed CD117) and the #1.1 was directed to the perivascular lineage. The injury (contusion) was performed at T8 level by mean of the Infinite Horizon Device, that allows the generation of reproducible traumatic lesion to the cord. The animals were divided into four groups: 1) lesioned mice transplanted with PBS (control group), 2) lesioned mice transplanted with cells from the #1.1 culture, 3) lesioned mice transplanted with cells from the #3.5 culture and 4) lesioned mice transplanted with cells from the #3.6 culture. All the transplanted cells, injected intravenously one week after the lesion, were labelled using Quantum dot (Invitrogen). The hindlimb motor recovery was evaluated for the 35 days following the lesion according to the Basso Mouse Scale (6). From 14 days after transplantation, the cell cultures #3.6 and #3.5 induced a better recovery of motor function than PBS, whereas cell culture #1.1 failed. Spinal cord transversal sections were analyzed for the presence of Qdot labelled transplanted AF cells and for the expression of neural-glial markers. One month after i.v. transplantation several Qdot positive cells at the site of injury were found and some of these Qdot labelled cells were also positives for β-tubulin III and GFAP immunostaining. Furthermore, the histological analysis of the lesion site showed that animals transplanted with culture #3.6 had a better preserved ventral myelin, and the number of infiltrated inflammatory cells (macrophages and neutrophils) was also reduced in comparison to the control PBS treated animals. Transplanted cells were detected at the lesion site and 4 mm away in rostral position to the injured area. In a complementary set of experiments we transplanted AF cells into a spinal cord which was subjected to the hemisection at T8 level. Animals were divided into three groups: 1) lesioned mice injected with PBS (control group), 2) lesioned mice treated with intraspinal administration of AF cells (#3.6 culture) embedded in hydrogel and 3) lesioned mice treated with intraspinal administration of hydrogel alone. At one week after injury AF cells-hydrogel treated mice showed a better motor recovery compared to hydrogel and PBS treated mice, and the difference was very significant at 28 days. The hydrogel treated mice also presented a slight motor improvement than PBS group, but this increase was lower than that induced by AF cells-hydrogel treatment. Confocal analysis of transversal sections revealed the presence of Qdot positive cells surrounding the lesion site, indicating that AF cells migrated out of hydrogel and survived over one month after transplantation. These data imply that the administration of amniotic fluid derived cells with a muscle-neural phenotype can attenuate secondary degeneration following traumatic lesion to spinal cord by dropping the cellular component of neuroinflammatory reaction. References: 1. Reier PJ (2004) NeuroRx 1:424-451 2. Schultz SS (2005) Curr Drug Targets 6:63-73 3. Gosden CM (1983) British Medical Bullettin 39(4):348-354 4. Prusa AR et al. (2004) Am J Obstet Gynecol 191(1) 309-314 5. De Coppi P et al. (2007) Nature Biotecnology 25(1):100-105 6. Basso DM et al. (2006) J Neurotrauma 23:635
14-ott-2009
Amniotic fluid stem cells; spinal cord injury; transplantation
Settore BIO/14 - Farmacologia
Settore BIO/09 - Fisiologia
Settore BIO/11 - Biologia Molecolare
Settore BIO/13 - Biologia Applicata
Third trimester human amniotic fluid cells with potential for therapy in neurodegenerative disorders / D. Cigognini, E. Nicora, E. Ripamonti, R. Adami, M. Menarini, A.M. Di Giulio, D. Bottai, A. Gorio. ((Intervento presentato al 34. convegno Congresso Nazionale della Società Italiana di Farmacologia tenutosi a Rimini nel 2009.
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