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Stem cells for nerve repair and prevention of muscle atrophy
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

  Peripheral nerve injury (PNI) is common and despite modern microsurgical techniques of repair, functional restoration is always incomplete. This results in impaired sensation and reduced motor function alongside pain and cold intolerance. Traumatic PNI are often associated with loss of nerve tissue, creating a gap, and direct repair of the two damaged nerve stumps is not possible. These types of injuries are reconstructed using autologous nerve grafts but this is far from ideal since it necessitates the sacrifice of a functional nerve from elsewhere in the body. Chronic muscle atrophy because of the prolonged delay in nerve regeneration across gaps is a significant impediment to an optimal functional recovery.

  Tissue engineering and regenerative medicine approaches to nerve repair might one day replace the need for autologous nerve grafts. This thesis investigates the effects of adipose derived stem cells (ASC) on nerve regeneration and muscle recovery by using the stem cells for intramuscular injection and combined with a biomaterial, poly-3-hydroxybutyrate (PHB), to create a bioengineered artificial nerve repair construct.  The mechanisms of interaction between the stem cells and neuromuscular system cells were investigated and with a view to translating this work into clinical practice, an optimal source of cells was investigated from human donors.

  It was hypothesized that injecting regenerative cells into muscle would reduce nerve injury induced muscle atrophy. A rat sciatic nerve lesion was performed and three different types of cells were injected into the denervated gastrocnemius muscle; either (1) undifferentiated ASC, (2) ASC induced to a ‘Schwann cell-like’ phenotype (dASC) or (3) primary Schwann cells. Nerves were either repaired by direct end-end suture or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology. The Schwann cells and dASC groups showed significantly better scores on functional tests when compared with control injections of growth medium alone. Muscle weight and histology were also significantly improved in the cell groups in comparison with the control group.

  PHB strips seeded with either primary Schwann cells or dASC suspended in a fibrin glue matrix were used to bridge a 10mm rat sciatic nerve gap. After 12 weeks, functional and morphological analysis (walking track test, electromyography, muscle weight and muscle and nerve histology) was performed. The results showed significantly better functional results for the PHB strips seeded with cells versus the control group with fibrin matrix only. This correlated with less muscle atrophy and greater distal axon myelination in the cell groups.

  To further optimize the nerve regeneration and muscle recovery, the nerve gap lesion was repaired by treatment with the bioengineered constructs seeded with dASC or nerve autograft in combination with stem cell injection in the muscle. After 6 weeks, the best results were obtained in the nerve graft group combined with intramuscular dASC injection which showed significantly less atrophy than the other groups. The results also showed that using the stem cells in a matrix on a PHB strip in combination with intramuscular injections could significantly reduce muscle atrophy.

  In vitro experiments showed that dASC expressed a wide range of neurotrophic and myogenic factors including BDNF, VEGF-A, IGF-1 and HGF. Stem cell conditioned medium enhanced the proliferation of myoblast cell lines and primary Schwann cells. Various signaling pathways (PKA, MAP kinase) were involved in these effects dependent on the cell type investigated. Furthermore, in direct co-culture with myoblast cells, a small population of the cells fused together to form myotube-like structures and expressed myogenic markers.

  Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  Cells from the superficial layer proliferated significantly faster than those from the deep layer. Superficial layer ASC induced significantly enhanced neurite outgrowth from neuronal cell lines when compared with the deep layer cells.  However, RT-PCR and ELISA analysis showed that ASC isolated from both layers expressed similar levels of the neurotrophic factors NGF, BDNF and GDNF.

  In summary, these results show that stem cell therapy at both levels (the nerve lesion site and in the target denervated muscle) offers a promising approach for clinical application for treatment of peripheral nerve lesions. The bioengineered artificial nerve construct, combining PHB strip with cells, also provides a beneficial environment for nerve regeneration. Many of the benefits of the ASC are likely to be mediated through their secretome, a rich source of neurotrophic and myogenic factors. Thus adipose tissue contains a pool of regenerative stem cells which have significant potential application to tissue engineering and regenerative medicine for nerve repair.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2015. , 46 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1726
Keyword [en]
adipose stem cells, biomaterial, muscle, nerve injury, neuromuscular junction, regeneration
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
cellforskning; Medical Cell Biology
Identifiers
URN: urn:nbn:se:umu:diva-106907ISBN: 978-91-7601-310-6 (print)OAI: oai:DiVA.org:umu-106907DiVA: diva2:845710
Public defence
2015-09-08, KB3A9, KBC Huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-08-18 Created: 2015-08-12 Last updated: 2015-08-18Bibliographically approved
List of papers
1. Regenerative cell injection in denervated muscle reduces atrophy and enhances recovery following nerve repair
Open this publication in new window or tab >>Regenerative cell injection in denervated muscle reduces atrophy and enhances recovery following nerve repair
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2013 (English)In: Muscle and Nerve, ISSN 0148-639X, E-ISSN 1097-4598, Vol. 47, no 5, 691-701 p.Article in journal (Refereed) Published
Abstract [en]

Introduction: Functional muscle recovery after peripheral nerve injury is far from optimal, partly due to atrophy of the muscle arising from prolonged denervation. We hypothesized that injecting regenerative cells into denervated muscle would reduce this atrophy. Methods: A rat sciatic nerve lesion was performed, and Schwann cells or adipose-derived stem cells, untreated or induced to a Schwann-celllike phenotype (dASC), were injected into the gastrocnemius muscle. Nerves were either repaired immediately or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology. Results: Schwann cells and dASC groups showed significantly better scores on functional tests when compared with injections of growth medium alone. Muscle weight and histology were also significantly improved in these groups. Conclusion: Cell injections may reduce muscle atrophy and could benefit nerve injury patients.

Keyword
denervation, muscle atrophy, peripheral nerve lesion, Schwann cells, stem cells
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-72702 (URN)10.1002/mus.23662 (DOI)000318234200010 ()
Available from: 2013-06-11 Created: 2013-06-10 Last updated: 2017-12-06Bibliographically approved
2. Poly-3-hydroxybutyrate strips seeded with regenerative cells are effective promoters of peripheral nerve repair
Open this publication in new window or tab >>Poly-3-hydroxybutyrate strips seeded with regenerative cells are effective promoters of peripheral nerve repair
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2017 (English)In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005, Vol. 11, no 3, 812-821 p.Article in journal (Refereed) Published
Abstract [en]

Peripheral nerve injuries are often associated with loss of nerve tissue and require a graft to bridge the gap. Autologous nerve grafts are still the 'gold standard' in reconstructive surgery but have several disadvantages, such as sacrifice of a functional nerve, neuroma formation and loss of sensation at the donor site. Bioengineered grafts represent a promising approach to address this problem. In this study, poly-3-hydroxybutyrate (PHB) strips were used to bridge a 10 mm rat sciatic nerve gap and their effects on long-term (12 weeks) nerve regeneration were compared. PHB strips were seeded with different cell types, either primary Schwann cells (SCs) or SC-like differentiated adipose-derived stem cells (dASCs) suspended in a fibrin glue matrix. The control group was PHB and fibrin matrix without cells. Functional and morphological properties of the regenerated nerve were assessed using walking track analysis, EMGs, muscle weight ratios and muscle and nerve histology. The animals treated with PHB strips seeded with SCs or dASCs showed significantly better functional ability than the control group. This correlated with less muscle atrophy and greater axon myelination in the cell groups. These findings suggest that the PHB strip seeded with cells provides a beneficial environment for nerve regeneration. Furthermore, dASCs, which are abundant and easily accessible, constitute an attractive cell source for future applications of cell therapy for the clinical repair of traumatic nerve injuries.

Keyword
biomaterial, muscle, nerve injury, regeneration, Schwann cells, stem cells
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-106901 (URN)10.1002/term.1980 (DOI)000398046600021 ()25556632 (PubMedID)
Available from: 2015-08-12 Created: 2015-08-12 Last updated: 2017-04-20Bibliographically approved
3. A comparison of an artificial nerve repair construct and nerve grafting when used in combination with intramuscular injections of stem cells for reduction of muscle atrophy
Open this publication in new window or tab >>A comparison of an artificial nerve repair construct and nerve grafting when used in combination with intramuscular injections of stem cells for reduction of muscle atrophy
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Peripheral nerve injuries represent a clinical challenge, especially when they are accompanied by loss of neural tissue. We tried to attain a better outcome after a peripheral nerve injury (in a rat sciatic nerve experimental model) by both repairing the nerve lesion and treating the denervated muscle at the same time.  We compared artificial nerve constructs made from strips of poly-3-hydroxybutyrate (PHB), seeded with or without Schwann cell-like differentiated adipose stem cells (dASC), and autografts (reverse sciatic nerve grafts) in combination with stem cell injections in the gastrocnemius muscle. dASC expressed Schwann cell markers (GFAP, S100B and p75-NTR) and secreted a range of nerve, angiogenic and muscle growth factors. Six weeks after nerve injury, the effects of the stem cells on nerve regeneration and reduction of muscle atrophy were assessed. PHB strips showed a high number of βIII-tubulin positive axons entering the distal stump and abundant endothelial cells. Animals treated with PHB strips without cells in combination with control growth medium intramuscular injections showed significantly more atrophy than the other groups.  Best results were obtained in the autograft group combined with intramuscular stem cell injections. This bioengineering research is a promising approach to treat nerve lesions and associated muscle atrophy.

Keyword
adipose-derived stem cells; muscle atrophy; peripheral nerve regeneration; poly-3-hydroxybutyrate
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-106889 (URN)
Available from: 2015-08-12 Created: 2015-08-12 Last updated: 2016-02-22Bibliographically approved
4. Mechanisms of adipose stem cell interactions with muscle cells and Schwann cells
Open this publication in new window or tab >>Mechanisms of adipose stem cell interactions with muscle cells and Schwann cells
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Peripheral nerve injury leads to muscle atrophy due to prolonged denervation. In a previous study, we showed the benefits of injecting Schwann cell and Schwann cell-like differentiated adipose stem cells (dASC) into the muscle to help nerve regeneration and prevent muscle atrophy. In this in vitro study, we have analyzed the possible mechanisms of how adipose stem cells interact with muscle cells and Schwann cells. Myoblast cell lines (C2C12 and L6) or rat primary Schwann cells treated with conditioned media prepared from either undifferentiated adipose stem cells or dASC proliferated more than control cultures. Stem cell mediated proliferation of myoblasts and Schwann cells was blocked by the inhibition of MAP kinase signaling (using U0126 drug) whereas the PKA pathway (inhibited with H89 drug) was only involved in myoblast proliferation. In order to assess the direct interaction of the stem cells with the muscle, we established direct in vitro co-culture of L6 myoblasts and stem cells for 2 weeks.  Under these conditions a small fraction of cells fused together forming multi-nucleated elongated structures, characteristic of myotubes.  These structures stained positive for fast type myosin heavy chain protein and myogenin. These effects were most pronounced in the dASC-myoblast co-cultures. ELISA analysis of the co-cultures showed high levels of secreted vascular endothelial growth factor-A (VEGF-A) and insulin like growth factor 1 proteins. Western blot analysis of denervated rat muscle tissue also showed elevated levels of VEGF-A expression in animals treated with stem cell injections. In conclusion, this study provides evidence of possible mechanisms how stem cells might influence cells of the neuromuscular system and supports the beneficial effect of using these cells for future clinical application in treatment of peripheral nerve injuries.

National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-106897 (URN)
Available from: 2015-08-12 Created: 2015-08-12 Last updated: 2016-02-22Bibliographically approved
5. Neurotrophic activity of human adipose stem cells isolated from deep and superficial layers of abdominal fat
Open this publication in new window or tab >>Neurotrophic activity of human adipose stem cells isolated from deep and superficial layers of abdominal fat
2011 (English)In: Cell and Tissue Research, ISSN 0302-766X, E-ISSN 1432-0878, Vol. 344, no 2, 251-260 p.Article in journal (Refereed) Published
Abstract [en]

New approaches to the clinical treatment of traumatic nerve injuries may one day utilize stem cells to enhance nerve regeneration. Adipose-derived stem cells (ASC) are found in abundant quantities and can be harvested by minimally invasive procedures that should facilitate their use in such regenerative applications. We have analyzed the properties of human ASC isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures. Cells from the superficial layer proliferate significantly faster than those from the deep layer. In both the deep and superficial layers, ASC express the pluripotent stem cell markers oct4 and nanog and also the stro-1 cell surface antigen. Superficial layer ASC induce the significantly enhanced outgrowth of neurite-like processes from neuronal cell lines when compared with that of deep layer cells. However, analysis by reverse transcription with the polymerase chain reaction and by enzyme-linked immunosorbent assay has revealed that ASC isolated from both layers express similar levels of the following neurotrophic factors: nerve growth factor, brain-derived neurotrophic factor and glial-derived neurotrophic factor. Thus, human ASC show promising potential for the treatment of traumatic nerve injuries. In particular, superficial layer ASC warrant further analysis of their neurotrophic molecules.

Keyword
Growth factor; Nerve; Regeneration; Stem cell; Tissue engineering
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-42254 (URN)10.1007/s00441-011-1142-5 (DOI)21400216 (PubMedID)
Available from: 2011-04-06 Created: 2011-04-06 Last updated: 2017-12-11Bibliographically approved

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