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Schwann cells and mesenchymal stem cells as promoter of peripheral nerve regeneration
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.
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The transplantation of primary Schwann cells (SC) has been shown to improve nerve regeneration. However, to monitor the survival of transplanted cells within the host, a stable labelling method is required. The in vitro characteristics of green fluorescent protein labelled SC (GFP SC) and their effects in an in vivo peripheral nerve injury model were investigated.   The GFP-SC were readily visualised ex vivo and stimulated significantly better axonal regeneration compared to controls. Clinical use of autologous SC for the treatment of nerve injuries is of limited use due to difficulty in obtaining clinically useful numbers. However, bone marrow mesenchymal stem cells (MSC) can trans-differentiate into SC like cells (dMSC). The in vitro and in vivo differentiation of MSC was explored, and the study extended to include the easily-accessible adipose stem cells (ASC).  In vitro, glial growth factor stimulated MSC express S100, a SC marker, and its expression is maintained following in vivo transplantation.  Similarly, untreated MSC transplanted in vivo also expressed S100, which indicates glial differentiation in response to local cytokines and growth factors. Using an in vitro model, comprising dMSC or dASC co-cultured with adult dorsal root ganglia (DRG) neurons, the capacity of the dMSC and SC like differentiated ASC (dASC) to promote axon myelination was verified: both cell types expressed transcripts for protein zero, peripheral myelin protein-22 and myelin basic protein.

The potential of stem cells in nerve repair may be limited by innate cellular senescence or donor age affecting cell functionality thus it was essential to determine the effects of donor age on morphology and functionality of stem cells.  The proliferation rates, expression of senescence markers (p38 and p53) and the stimulation of neurite outgrowth from DRG neurons by stem cells isolated from neonatal, young or old rats were very similar. However, the distribution and ultrastructure of mitochondria in dMSC and dASC from young and old rats were quite different, and seem to indicate physiological senescence of the aged cells.  Given the wide-ranging influence of Notch signalling in cell differentiation, including the neural crest to a glial cell type switch, and self-renewal in mammals, its role in the differentiation of stem cells to SC was investigated. The mRNA for notch-1 and -2 receptors were expressed in the dASC, blockage of notch signaling did not affect the neurotrophic and myelination potential of dASC. 

In conclusion, these findings show that GFP labelling has no deleterious effect on SC survival and function. MSC and ASC differentiated into glial-type cells acquire SC morphology, and express characteristic SC markers, and the differentiation process was independent of the Notch signaling pathway. Also, following transplantation into a nerve gap injury dMSC improve regeneration. This study established that following co-culture with DRG neurons, dMSC and dASC were able to express peripheral myelin proteins.  Also, the functional bioactivity of these cells is independent of the donor animal age. Finally, although the glial lineage differentiated aged cells characterized in this study expressed markers typical of senescence they retained the potential to support axon regeneration.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2011. , 92 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1434
Keyword [en]
peripheral nerve regeneration, Schwann cell, adult stem cell, myelin, senescence
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-48413ISBN: 978-91-7459-277-1 (print)OAI: oai:DiVA.org:umu-48413DiVA: diva2:449102
Public defence
2011-11-24, sal BiA201, Biologihuset, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2011-11-03 Created: 2011-10-19 Last updated: 2012-11-09Bibliographically approved
List of papers
1. Green fluorescent protein is a stable morphological marker for schwann cell transplants in bioengineered nerve conduits
Open this publication in new window or tab >>Green fluorescent protein is a stable morphological marker for schwann cell transplants in bioengineered nerve conduits
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2004 (English)In: Tissue engineering, ISSN 1076-3279, E-ISSN 1557-8690, Vol. 10, no 9-10, 1359-1367 p.Article in journal (Refereed) Published
Abstract [en]

Bioengineered systems incorporate cultured cells to mimic the substituted tissue. A labeling method is necessary to monitor the survival of transplanted cells within the host. This labeling method must be compatible with the histochemical methods used for morphological analysis. This study assessed (1) The in vitro characteristics of Schwann cells (SCs) labeled with green fluorescent protein (GFP), (2) the in vivo effect of transplanted GFP-SCs in a model of peripheral nerve injury, and (3) the compatibility of GFP-SCs with immunofluorescence histochemical techniques. SCs were retrovirally labeled with GFP and their growth characteristics were compared with those of nontransduced SCs (ntSCs). GFP-SCs were seeded in a resorbable nerve conduit for grafting into a 1-cm gap in rat sciatic nerve. Grafts were harvested after 2 weeks and immunofluorescent staining was performed to measure axonal and SC regeneration distances and to identify GFP-SCs. Results of GFP-SC vitality assays did not vary significantly from those of ntSC assays. GFP-SCs were readily located ex vivo and stimulated significantly better axonal and SC regeneration distances in comparison with empty conduits. These findings show that GFP labeling does not have a deleterious effect on SCs and that it is a useful labeling method for the study of bioengineered systems.

Place, publisher, year, edition, pages
Larchmont, NY: Mary Ann Liebert, Inc., 2004
Keyword
Animals, Animals, Newborn, Biological Markers/metabolism, Biomedical Engineering/methods, Cell Survival/*physiology, Cells; Cultured, Green Fluorescent Proteins/genetics/*metabolism, Microscopy; Fluorescence/methods, Nerve Regeneration/physiology, Rats, Rats; Sprague-Dawley, Recombinant Proteins/metabolism, Schwann Cells/metabolism/*pathology/*transplantation, Sciatic Nerve/injuries/metabolism/*pathology/*surgery, Tissue Engineering/*methods, Transplants
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-6412 (URN)10.1089/ten.2004.10.1359 (DOI)15588396 (PubMedID)
Available from: 2007-12-11 Created: 2007-12-11 Last updated: 2017-12-14Bibliographically approved
2. Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration
Open this publication in new window or tab >>Rat bone marrow mesenchymal stem cells express glial markers and stimulate nerve regeneration
2004 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 362, no 3, 200-203 p.Article in journal (Refereed) Published
Abstract [en]

Bone marrow mesenchymal stem cells can trans-differentiate into neuronal phenotypes. We examined the differentiation of marrow stromal cells (MSCs) in culture and during nerve regeneration. MSCs from adult rats were exposed to glial growth factor (GGF) to stimulate glial differentiation. Subsequently differentiated MSCs were retrovirally labelled with green fluorescent protein and transplanted into 1 cm nerve conduits in the rat sciatic nerve. Fifteen days post-operatively the conduits were examined for axonal and Schwann cell regeneration and MSC integration. In vitro, MSCs exposed to GGF expressed S100 and glial fibrillary acidic protein. Following transplantation, MSCs maintained S100 expression and enhanced nerve regeneration, with significant Schwann cell regeneration compared to control (2.7 +/- 0.21 vs. 2.05 +/- .21 mm; P < 0.05). MSCs not exposed to GGF prior to transplantation expressed S100 in vivo indicating glial differentiation in response to local cytokines and growth factors.

Place, publisher, year, edition, pages
Elsevier, 2004
Keyword
Animals, Bone Marrow Cells/cytology/drug effects/metabolism/*physiology, Bone Marrow Transplantation/methods, Cell Count/methods, Cell Differentiation/drug effects/*physiology, Gene Expression Regulation, Glial Fibrillary Acidic Protein/*metabolism, Green Fluorescent Proteins, Immunohistochemistry/methods, Luminescent Proteins/metabolism, Nerve Regeneration/*physiology, Neuregulin-1/metabolism, Rats, Rats; Sprague-Dawley, Retroviridae/metabolism, S100 Proteins/*metabolism, Sciatic Nerve/transplantation, Stromal Cells/drug effects/metabolism
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-12711 (URN)10.1016/j.neulet.2004.03.077 (DOI)15158014 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2017-12-14Bibliographically approved
3. Bone marrow- and adipose-derived stem cells show expression of myelin mRNAs and proteins
Open this publication in new window or tab >>Bone marrow- and adipose-derived stem cells show expression of myelin mRNAs and proteins
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2010 (English)In: Regenerative medicine, ISSN 1746-076X, Vol. 5, no 3, 403-410 p.Article in journal (Refereed) Published
Abstract [en]

Aims: PNS myelin is formed by Schwann cells (SCs). In this study, we applied an in vitro model to study myelin formation, using bone marrow mesenchymal stem cells and adipose-derived stem cells differentiated into SC-like cells and co-cultured with dissociated adult dorsal root ganglia neurons.

Methods: Immunocytochemistry, reverse transcription-PCR and western blotting techniques were used to investigate the expression of myelin proteins at both the transcriptional and translational level.

Results: Transcripts for protein zero, peripheral myelin protein 22 and myelin basic protein were detected in differentiated stem cells following co-culture with neuronal cells. Furthermore, protein zero, peripheral myelin protein 22 and myelin basic proteins were recognized in the co-cultures. These results were consistent with immunostaining of myelin proteins and with observation by electron microscopy.

Conclusion: Both types of adult stems cells differentiated into SC-like cells have potential to myelinate neuronal cells during regeneration, being functionally identical to SCs of the PNS.

Keyword
adipose, mesenchymal, myelin, stem cells
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-41349 (URN)10.2217/rme.10.15 (DOI)20455651 (PubMedID)
Available from: 2011-03-23 Created: 2011-03-23 Last updated: 2011-11-03Bibliographically approved
4. Notch independent signalling mediates Schwann cell-like differentiation of adipose derived stem cells
Open this publication in new window or tab >>Notch independent signalling mediates Schwann cell-like differentiation of adipose derived stem cells
2009 (English)In: Neuroscience Letters, ISSN 0304-3940, E-ISSN 1872-7972, Vol. 467, no 2, 164-168 p.Article in journal (Refereed) Published
Abstract [en]

Adipose derived stem cells (ASC) differentiate into a Schwann cell (SC)-like phenotype but the signalling pathways mediating this are unknown. We hypothesised that notch might be involved, given its important role in regulating SC development. Rat ASC were differentiated using bFGF, PDGF, GGF-2 and forskolin. RT-PCR analysis showed that mRNA for notch-1 and notch-2 receptors and the notch responsive gene, hes-1, were expressed throughout the differentiation process whereas jagged-1 a notch ligand, and the hey-1 gene were markedly down-regulated. In contrast delta-1 was up-regulated with differentiation and was strongly expressed by rat primary SC. Treatment of ASC with N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase inhibitor which blocks notch signalling, had no effect on up-regulation of SC proteins S100 or GFAP during differentiation. Furthermore, when co-cultured with NG108-15 neurons, differentiated ASC cultures treated in the absence or presence of DAPT enhanced neurite outgrowth to similar levels. Differentiated ASC expressed PMP-22 but P0 was only present when co-cultured with dorsal root ganglia neurons. DAPT did not affect the expression of these myelin proteins. Thus, ASC express components of the notch signalling pathway but our studies suggest notch is unlikely to play a role in the neurotrophic activity and myelination capability of ASC differentiated into SC-like cells.

Place, publisher, year, edition, pages
Elsevier, 2009
Keyword
Glia, Myelination, Neurotrophic, Peripheral nerve
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-48410 (URN)10.1016/j.neulet.2009.10.030 (DOI)
Available from: 2011-10-27 Created: 2011-10-19 Last updated: 2017-12-08Bibliographically approved
5. Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages
Open this publication in new window or tab >>Morphological, molecular and functional differences of adult bone marrow- and adipose-derived stem cells isolated from rats of different ages
Show others...
2012 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 318, no 16, 2034-2048 p.Article in journal (Refereed) Published
Abstract [en]

Adult mesenchymal stem cells have self-renewal and multiple differentiation potentials, and play important roles in regenerative medicine. However, their use may be limited by senescence or age of the donor, leading to changes in stem cell functionality. We investigated morphological, molecular and functional differences between bone marrow-derived (MSC) and adipose-derived (ASC) stem cells isolated from neonatal, young and old rats compared to Schwann cells from the same animals. Immunocytochemistry, RT-PCR, proliferation assays, western blotting and transmission electron microscopy were used to investigate expression of senescence markers. Undifferentiated and differentiated ASC and MSC from animals of different ages expressed Notch-2 at similar levels; protein-38 and protein-53 were present in all groups of cells with a trend towards increased levels in cells from older animals compared to those from neonatal and young rats. Following co-culture with adult neuronal cells, dMSC and dASC from animals of all ages elicited robust neurite outgrowth. Mitotracker (R) staining was consistent with ultrastructural changes seen in the mitochondria of cells from old rats, indicative of senescence. In conclusion, this study showed that although the cells from aged animals expressed markers of senescence, aged MSC and ASC differentiated into SC-like cells still retain potential to support axon regeneration.

Keyword
Strol, Notch, p53, p38, Mitochondria, Senescence, Aging
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-61175 (URN)10.1016/j.yexcr.2012.05.008 (DOI)000307210100008 ()
Note

Included in Cristina Mantovanis thesis in manuscript form.

Available from: 2012-11-09 Created: 2012-11-07 Last updated: 2017-12-07Bibliographically approved

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