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Mesenchymal stem cells for repair of the peripheral and central nervous system
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. (Neurogruppen)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Odlade mesenkymala stamcellers användning vid skador på perifera och centrala nervsystemet (Swedish)
Abstract [en]

Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord.

Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF).

Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons.

Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine.

After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn.

In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2011. , 59 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1433
Keyword [en]
Bone marrow-derived stromal cells, Schwann cells, Peripheral nerve injury, Spinal cord injury, Neurotransplantation
National Category
Neurosciences
Research subject
Human Anatomy; cellforskning
Identifiers
URN: urn:nbn:se:umu:diva-47746ISBN: 978-91-7459-240-5 (print)OAI: oai:DiVA.org:umu-47746DiVA: diva2:444268
Public defence
2011-10-20, BiA201, Biologihuset, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-09-29 Created: 2011-09-28 Last updated: 2011-09-29Bibliographically approved
List of papers
1. Characterisation of human mesenchymal stem cells following differentiation into Schwann cell-like cells
Open this publication in new window or tab >>Characterisation of human mesenchymal stem cells following differentiation into Schwann cell-like cells
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2009 (English)In: Neuroscience research, ISSN 0168-0102, E-ISSN 1872-8111, Vol. 64, no 1, 41-49 p.Article in journal (Refereed) Published
Abstract [en]

Cell-based therapies provide a clinically applicable and available alternative to nerve autografts. Our previous studies have characterised rat-derived mesenchymal stem cells (MSC) and here we have investigated the phenotypic, molecular and functional characteristics of human-derived MSC (hMSC) differentiated along a Schwann cell lineage. The hMSC were isolated from healthy human donors and the identity of the undifferentiated hMSC was confirmed by the detection of MSC specific cells surface markers. The hMSC were differentiated along a glial cell lineage using an established cocktail of growth factors including glial growth factor-2. Following differentiation, the hMSC expressed the key Schwann cell (SC) markers at both the transcriptional and translational level. More importantly, we show the functional effect of hMSC on neurite outgrowth using an in vitro co-culture model system with rat-derived primary sensory neurons. The number of DRG sprouting neurites was significantly enhanced in the presence of differentiated hMSC; neurite length and density (branching) were also increased. These results provide evidence that hMSC can undergo molecular, morphological and functional changes to adopt a SC-like behaviour and, therefore, could be suitable as SC substitutes for nerve repair in clinical applications.

Keyword
Bone marrow stromal cell; Schwann cell; Glial cell; Differentiation; Dorsal root ganglion; Glial growth factor
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-24123 (URN)10.1016/j.neures.2009.01.010 (DOI)19428682 (PubMedID)
Available from: 2009-06-30 Created: 2009-06-30 Last updated: 2017-12-13Bibliographically approved
2. Aging effect on neurotrophic activity of human mesenchymal stem cells
Open this publication in new window or tab >>Aging effect on neurotrophic activity of human mesenchymal stem cells
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 9, e45052- p.Article in journal (Refereed) Published
Abstract [en]

Clinical efficacy of stem cells for nerve repair is likely to be influenced by issues including donor age and in vitro expansion time. We isolated human mesenchymal stem cells (MSC) from bone marrow of young (16–18 years) and old (67–75 years) donors and analyzed their capacity to differentiate and promote neurite outgrowth from dorsal root ganglia (DRG) neurons. Treatment of MSC with growth factors (forskolin, basic fibroblast growth factor, platelet derived growth factor-AA and glial growth factor-2) induced protein expression of the glial cell marker S100 in cultures from young but not old donors. MSC expressed various neurotrophic factor mRNA transcripts. Growth factor treatment enhanced the levels of BDNF and VEGF transcripts with corresponding increases in protein release in both donor cell groups. MSC in co-culture with DRG neurons significantly enhanced total neurite length which, in the case of young but not old donors, was further potentiated by treatment of the MSC with the growth factors. Stem cells from young donors maintained their proliferation rate over a time course of 9 weeks whereas those from the old donors showed increased population doubling times. MSC from young donors, differentiated with growth factors after long-term culture, maintained their ability to enhance neurite outgrowth of DRG. Therefore, MSC isolated from young donors are likely to be a favourable cell source for nerve repair.

Place, publisher, year, edition, pages
San Francisco: Public Library of Science, 2012
Keyword
adult stem cell, glia, nerve injury, regeneration
National Category
Neurosciences
Research subject
Human Anatomy; cellforskning
Identifiers
urn:nbn:se:umu:diva-47755 (URN)10.1371/journal.pone.0045052 (DOI)000309742800031 ()
Available from: 2011-09-28 Created: 2011-09-28 Last updated: 2017-12-08Bibliographically approved
3. Fibrin conduit supplemented with human mesenchymal stem cells supports regeneration after peripheral nerve injury   
Open this publication in new window or tab >>Fibrin conduit supplemented with human mesenchymal stem cells supports regeneration after peripheral nerve injury   
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

To address the need for the development of bioengineered replacement of a nerve graft for treatment of peripheral nerve injuries a novel two component fibrin glue conduit was combined with human mesenchymal stem cells (hMSC) and immunosupressive treatment with cyclosporine. MSC possess the advantage of lower donor site morbidity and easier expandability in vitro compared with Schwann cells. The effects of hMSC on axonal regeneration in the conduit and reaction of activated macrophages was investigated using sciatic nerve injury model. The experiments were performed on 20 female Fischer rats (8-10 weeks old). A 10mm gap in the sciatic nerve was created and repaired either with fibrin glue conduit containing diluted fibrin matrix or fibrin glue conduit containing fibrin matrix with hMSC at concentration of 80x106 cells per ml. Cells were labeled with PKH26 prior to transplantation. The animals were allowed to survive for 3 weeks and some groups were treated with daily injections of cyclosporine. After 3 weeks the conduits were harvested and the distance of regeneration and area occupied by regenerating axons together with ED1 staining of activated macrophages was measured. hMSC survived in the conduit and enhanced axonal regeneration only when transplantation was combined with cyclosporine treatment. Moreover, cyclosporine significantly reduced the ED1 macrophage reaction.

Keyword
Peripheral nerve injury, Nerve conduit, Bone marrow, Mesenchymal stem cells, Regeneration
National Category
Neurosciences
Research subject
Human Anatomy; cellforskning
Identifiers
urn:nbn:se:umu:diva-47756 (URN)
Available from: 2011-09-28 Created: 2011-09-28 Last updated: 2011-09-29Bibliographically approved
4. Neuroprotective and growth-promoting effects of bone marrow stromal cells after cervical spinal cord injury in adult rats
Open this publication in new window or tab >>Neuroprotective and growth-promoting effects of bone marrow stromal cells after cervical spinal cord injury in adult rats
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2011 (English)In: Cytotherapy, ISSN 1465-3249, E-ISSN 1477-2566, Vol. 13, no 7, 873-887 p.Article in journal (Refereed) Published
Abstract [en]

Background aims. Bone marrow stromal cells (BMSC) have been shown to provide neuroprotection after transplantation into the injured central nervous system. The present study investigated whether adult rat BMSC differentiated along a Schwann cell lineage could increase production of trophic factors and support neuronal survival and axonal regeneration after transplantation into the injured spinal cord.

Methods. After cervical C4 hemi-section, 5-bromo-2-deoxyuridine (BrdU)/green fluorescent protein (GFP)-labeled BMSC were injected into the lateral funiculus at 1 mm rostral and caudal to the lesion site. Spinal cords were analyzed 2-13 weeks after transplantation.

Results and Conclusions. Treatment of native BMSC with Schwann cell-differentiating factors significantly increased production of brain-derived neurotrophic factor in vitro. Transplanted undifferentiated and differentiated BMSC remained at the injection sites, and in the trauma zone were often associated with neurofilament-positive fibers and increased levels of vascular endothelial growth factor. BMSC promoted extensive in-growth of serotonin-positive raphaespinal axons and calcitonin gene-related peptide (CGRP)-positive dorsal root sensory axons into the trauma zone, and significantly attenuated astroglial and microglial cell reactions, but induced aberrant sprouting of CGRP-immunoreactive axons in Rexed's lamina III. Differentiated BMSC provided neuroprotection for axotomized rubrospinal neurons and increased the density of rubrospinal axons in the dorsolateral funiculus rostral to the injury site. The present results suggest that BMSC induced along the Schwann cell lineage increase expression of trophic factors and have neuroprotective and growth-promoting effects after spinal cord injury.

Keyword
bone marrow, mesenchymal stromal cells, red nucleus, retrograde degeneration, spinal cord trauma, transplantation
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-44220 (URN)10.3109/14653249.2011.574116 (DOI)21521004 (PubMedID)
Available from: 2011-05-26 Created: 2011-05-26 Last updated: 2017-12-11Bibliographically approved

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