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Transplantation of mesenchymal stem cells and injections of microRNA as therapeutics for nervous system repair
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Hand Surgery. Department of Surgical and Perioperative Sciences, Faculty of Medicine.
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Traumatic injuries to the spinal cord (SCI) and peripheral nerve (PNI) affect several thousand people worldwide every year. At present, there is no effective treatment for SCI and despite continuous improvements in microsurgical reconstructive techniques for PNI, many patients are still left with permanent, devastating neurological dysfunction. This thesis investigates the effects of mesenchymal stem cells (MSC) derived from adipose (ASC) and dental (DSC) tissue and chitosan/microRNA-124 polyplex particles on regeneration after spinal cord and peripheral nerve injury in adult rats. Dental stem cells were obtained from apical papilla, dental pulp, and periodontal ligament. ASC and DSC expressed MSC surface markers (CD73, CD90, CD105 and CD146) and various neurotrophic molecules including BDNF, GDNF, NGF, VEGF-A and angiopoietin-1. Growth factor stimulation of the stem cells resulted in increased secretion of these proteins. Both ASC and DSC supported in vitro neurite outgrowth and in contrast to Schwann cells, ASC did not induce activation of astrocytes. Stimulated ASC also showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. In a peripheral nerve injury model, ASC and DSC were seeded into a fibrin conduit, which was used to bridge a 10 mm rat sciatic nerve gap. After 2 weeks, both ASC and DSC promoted axonal regeneration in the conduit and reduced caspase-3 expression in the dorsal root ganglion (DRG). ASC also enhanced GAP-43 and ATF-3 expression in the spinal cord, reduced c-jun expression in the DRG and increased the vascularity of the implant. After transplantation into injured C3-C4 cervical spinal cord, ASC continued to express neurotrophic factors and laminin and stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure and the density of the astroglial scar. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the reactivity of OX42-positive microglial cells was markedly reduced. However, ASC did not enhance recovery of forelimb function. In order to reduce activation of microglia/macrophages and the secondary tissue damage after SCI, the role of microRNA-124 was investigated. In vitro transfection of chitosan/microRNA-124 polyplex particles into rat microglia resulted in the reduction of reactive oxygen species and TNF-α levels and lowered expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia. Alternatively, particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury. Microinjections of chitosan/microRNA-124 particles significantly reduced the number of ED-1 positive macrophages after SCI. In summary, these results show that human MSC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for neural regeneration after spinal cord and peripheral nerve injury. The data also suggests that chitosan/microRNA-124 particles could be potential treatment technique to reduce neuroinflammation.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2016. , 97 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1802
Keyword [en]
spinal cord injury, peripheral nerve injury, mesenchymal stem cells, regeneration, neurotrophic factor, angiogenic factor
National Category
Neurosciences
Identifiers
URN: urn:nbn:se:umu:diva-119437ISBN: 978-91-7601-465-3 (print)OAI: oai:DiVA.org:umu-119437DiVA: diva2:920912
Public defence
2016-05-13, N360, Naturvetarhuset, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 2014-2306EU, European Research Council
Available from: 2016-04-22 Created: 2016-04-19 Last updated: 2016-05-26Bibliographically approved
List of papers
1. Stimulating the neurotrophic and angiogenic properties of human adipose-derived stem cells enhances nerve repair
Open this publication in new window or tab >>Stimulating the neurotrophic and angiogenic properties of human adipose-derived stem cells enhances nerve repair
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2014 (English)In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 23, no 7, 741-754 p.Article in journal (Refereed) Published
Abstract [en]

In future, adipose-derived stem cells (ASC) might be used to treat neurological disorders. In this study, the neurotrophic and angiogenic properties of human ASC were evaluated, and their effects in a peripheral nerve injury model were determined. In vitro growth factor stimulation of the cells resulted in increased secretion of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor-A (VEGF-A), and angiopoietin-1 proteins. Conditioned medium from stimulated cells increased neurite outgrowth of dorsal root ganglia (DRG) neurons. Similarly, stimulated cells showed an enhanced ability to induce capillary-like tube formation in an in vitro angiogenesis assay. ASC were seeded into a fibrin conduit that was used to bridge a 10 mm rat nerve gap. After 2 weeks, the animals treated with control or stimulated ASC showed an enhanced axon regeneration distance. Stimulated cells evoked more total axon growth. Analysis of regeneration and apoptosis-related gene expression showed that both ASC and stimulated ASC enhanced GAP-43 and activating transcription factor 3 (ATF-3) expression in the spinal cord and reduced c-jun expression in the DRG. Caspase-3 expression in the DRG was reduced by stimulated ASC. Both ASC and stimulated ASC also increased the vascularity of the fibrin nerve conduits. Thus, ASC produce functional neurotrophic and angiogenic factors, creating a more desirable microenvironment for nerve regeneration.

National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-83708 (URN)10.1089/scd.2013.0396 (DOI)000333613700007 ()24124760 (PubMedID)
Available from: 2013-12-05 Created: 2013-12-05 Last updated: 2017-12-06Bibliographically approved
2. The neurotrophic effects of different human dental mesenchymal stem cells
Open this publication in new window or tab >>The neurotrophic effects of different human dental mesenchymal stem cells
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, 12605Article in journal (Refereed) Published
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-119481 (URN)10.1038/s41598-017-12969-1 (DOI)000412138800041 ()28974767 (PubMedID)
Available from: 2016-04-20 Created: 2016-04-20 Last updated: 2017-11-20Bibliographically approved
3. The therapeutic effects of human adipose derived stem cells in a rat cervical spinal cord injury model
Open this publication in new window or tab >>The therapeutic effects of human adipose derived stem cells in a rat cervical spinal cord injury model
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2014 (English)In: Stem Cells and Development, ISSN 1547-3287, E-ISSN 1557-8534, Vol. 23, no 14, 1659-1674 p.Article in journal (Refereed) Published
Abstract [en]

Spinal cord injury triggers a cascade of degenerative changes leading to cell death and cavitation. Severed axons fail to regenerate across the scar tissue and are only capable of limited sprouting. In this study we investigated the effects of adult human adipose derived stem cells (ASC) on axonal regeneration following transplantation into the injured rat cervical spinal cord. ASC did not induce activation of astrocytes in culture and supported neurite outgrowth from adult rat sensory DRG neurons. After transplantation into the lateral funiculus 1mm rostral and caudal to the cervical C3-C4 hemisection, ASC continued to express BDNF, VEGF and FGF-2 for 3 weeks but only in animals treated with cyclosporine A. Transplanted ASC stimulated extensive ingrowth of 5HT-positive raphaespinal axons into the trauma zone with some terminal arborisations reaching the caudal spinal cord. In addition, ASC induced sprouting of raphaespinal terminals in C2 contralateral ventral horn and C6 ventral horn on both sides. Transplanted cells also changed the structure of the lesion scar with numerous astrocytic processes extended into the middle of the trauma zone in a chain-like pattern and in close association with regenerating axons. The density of the astrocytic network was also significantly decreased. Although the transplanted cells had no effect on the density of capillaries around the lesion site, the activity of OX42-positive microglial cells was markedly reduced. However, ASC did not support recovery of forelimb function. The results suggest that transplanted ASC can modify the structure of the glial scar and stimulate axonal sprouting.

Place, publisher, year, edition, pages
Mary Ann Liebert, 2014
National Category
Neurosciences Cell and Molecular Biology Hematology
Identifiers
urn:nbn:se:umu:diva-88637 (URN)10.1089/scd.2013.0416 (DOI)000339315000009 ()24803143 (PubMedID)
Note

Included in thesis in manuscript form.

Available from: 2014-05-12 Created: 2014-05-12 Last updated: 2017-12-05Bibliographically approved
4. Chitosan polyplex mediated delivery of miRNA-124 reduces activation of microglial cells in vitro and in rat models of spinal cord injury
Open this publication in new window or tab >>Chitosan polyplex mediated delivery of miRNA-124 reduces activation of microglial cells in vitro and in rat models of spinal cord injury
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2016 (English)In: Nanomedicine: Nanotechnology, Biology and Medicine, ISSN 1549-9634, E-ISSN 1549-9642, Vol. 12, no 3, 643-653 p.Article in journal (Refereed) Published
Abstract [en]

Traumatic injury to the central nervous system (CNS) is further complicated by an increase in secondary neuronal damage imposed by activated microglia/macrophages. MicroRNA-124 (miR-124) is responsible for mouse monocyte quiescence and reduction of their inflammatory cytokine production. We describe the formulation and ex vivo transfection of chitosan/miR-124 polyplex particles into rat microglia and the resulting reduction of reactive oxygen species (ROS) and TNF-α and lower expression of MHC-II. Upon microinjection into uninjured rat spinal cords, particles formed with Cy3-labeled control sequence RNA, were specifically internalized by OX42 positive macrophages and microglia cells. Alternatively particles injected in the peritoneum were transported by macrophages to the site of spinal cord injury 72h post injection. Microinjections of chitosan/miR-124 particles significantly reduced the number of ED-1 positive macrophages in the injured spinal cord. Taken together, these data present a potential treatment technique to reduce inflammation for a multitude of CNS neurodegenerative conditions.

National Category
Basic Medicine Nano Technology
Research subject
Human Anatomy
Identifiers
urn:nbn:se:umu:diva-117111 (URN)10.1016/j.nano.2015.10.011 (DOI)000373924000007 ()26582736 (PubMedID)
Note

2016-05-16: Originally published in manuscript form.

Available from: 2016-02-22 Created: 2016-02-22 Last updated: 2017-11-30Bibliographically approved

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