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El-Habta, R., Kingham, P. J. & Backman, L. J. (2018). Adipose stem cells enhance myoblast proliferation via acetylcholine and extracellular signal-regulated kinase 1/2 signaling. Muscle and Nerve, 57(2), 305-311
Open this publication in new window or tab >>Adipose stem cells enhance myoblast proliferation via acetylcholine and extracellular signal-regulated kinase 1/2 signaling
2018 (English)In: Muscle and Nerve, ISSN 0148-639X, E-ISSN 1097-4598, Vol. 57, no 2, p. 305-311Article in journal (Refereed) Published
Abstract [en]

Introduction: In this study we investigated the interaction between adipose tissue-derived stem cells (ASCs) and myoblasts in co-culture experiments. Methods: Specific inductive media were used to differentiate ASCs in vitro into a Schwann cell-like phenotype (differentiated adipose tissuederived stem cells, or dASCs) and, subsequently, the expression of acetylcholine (ACh)-related machinery was determined. In addition, the expression of muscarinic ACh receptors was examined in denervated rat gastrocnemius muscles. Results: In contrast to undifferentiated ASCs, dASCs expressed more choline acetyltransferase and vesicular acetylcholine transporter. When co-cultured with myoblasts, dASCs enhanced the proliferation rate, as did ACh administration alone. Western blotting and pharmacological inhibitor studies showed that phosphorylated extracellular signal-regulated kinase 1/2 signaling mediated these effects. In addition, denervated muscle showed higher expression of muscarinic ACh receptors than control muscle. Discussion: Our findings suggest that dASCs promote proliferation of myoblasts through paracrine secretion of ACh, which could explain some of their regenerative capacity in vivo.

Place, publisher, year, edition, pages
WILEY, 2018
Keywords
adipose-derived stem cells, denervation, myoblasts, nonneuronal acetylcholine, proliferation
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-144336 (URN)10.1002/mus.25741 (DOI)000419964700026 ()28686790 (PubMedID)
Available from: 2018-02-08 Created: 2018-02-08 Last updated: 2019-02-22Bibliographically approved
Jones, I., Yelhekar, T. D., Wiberg, R., Kingham, P. J., Johansson, S., Wiberg, M. & Carlsson, L. (2018). Development and validation of an in vitro model system to study peripheral sensory neuron development and injury. Scientific Reports, 8, Article ID 15961.
Open this publication in new window or tab >>Development and validation of an in vitro model system to study peripheral sensory neuron development and injury
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 15961Article in journal (Refereed) Published
Abstract [en]

The ability to discriminate between diverse types of sensation is mediated by heterogeneous populations of peripheral sensory neurons. Human peripheral sensory neurons are inaccessible for research and efforts to study their development and disease have been hampered by the availability of relevant model systems. The in vitro differentiation of peripheral sensory neurons from human embryonic stem cells therefore provides an attractive alternative since an unlimited source of biological material can be generated for studies that specifically address development and injury. The work presented in this study describes the derivation of peripheral sensory neurons from human embryonic stem cells using small molecule inhibitors. The differentiated neurons express canonical- and modality-specific peripheral sensory neuron markers with subsets exhibiting functional properties of human nociceptive neurons that include tetrodotoxin-resistant sodium currents and repetitive action potentials. Moreover, the derived cells associate with human donor Schwann cells and can be used as a model system to investigate the molecular mechanisms underlying neuronal death following peripheral nerve injury. The quick and efficient derivation of genetically diverse peripheral sensory neurons from human embryonic stem cells offers unlimited access to these specialised cell types and provides an invaluable in vitro model system for future studies.

Place, publisher, year, edition, pages
Nature Publishing Group, 2018
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-153701 (URN)10.1038/s41598-018-34280-3 (DOI)000448589200037 ()30374154 (PubMedID)
Funder
Swedish Research Council, 22292Gunvor och Josef Anérs stiftelseVästerbotten County Council
Available from: 2018-12-05 Created: 2018-12-05 Last updated: 2018-12-05Bibliographically approved
Wiberg, R., Novikova, L. N. & Kingham, P. J. (2018). Evaluation of apoptotic pathways in dorsal root ganglion neurons following peripheral nerve injury. NeuroReport, 779-785
Open this publication in new window or tab >>Evaluation of apoptotic pathways in dorsal root ganglion neurons following peripheral nerve injury
2018 (English)In: NeuroReport, ISSN 0959-4965, E-ISSN 1473-558X, p. 779-785Article in journal (Refereed) Published
Abstract [en]

Peripheral nerve injuries induce significant sensory neuronal cell death in the dorsal root ganglia (DRG); however, the role of specific apoptotic pathways is still unclear. In this study, we performed peripheral nerve transection on adult rats, after which the corresponding DRGs were harvested at 7, 14, and 28 days after injury for subsequent molecular analyses with quantitative reverse transcription-PCR, western blotting, and immunohistochemistry. Nerve injury led to increased levels of caspase-3 mRNA and active caspase-3 protein in the DRG. Increased expression of caspase-8, caspase-12, caspase-7, and calpain suggested that both the extrinsic and the endoplasmic reticulum (ER) stress-mediated apoptotic pathways were activated. Phosphorylation of protein kinase R-like ER kinase further implied the involvement of ER-stress in the DRG. Phosphorylated protein kinase R-like ER kinase was most commonly associated with isolectin B4 (IB4)-positive neurons in the DRG and this may provide an explanation for the increased susceptibility of these neurons to die following nerve injury, likely in part because of an activation of the ER-stress response.

Place, publisher, year, edition, pages
Lippincott Williams & Wilkins, 2018
National Category
Cell and Molecular Biology Neurosciences
Identifiers
urn:nbn:se:umu:diva-127356 (URN)10.1097/WNR.0000000000001031 (DOI)000433096700013 ()29659443 (PubMedID)
Note

Originally included in thesis in manuscript form.

Available from: 2016-11-09 Created: 2016-11-09 Last updated: 2018-06-25Bibliographically approved
McGrath, A. M., Brohlin, M., Wiberg, R., Kingham, P. J., Novikov, L. N., Wiberg, M. & Novikova, L. N. (2018). Long-Term Effects of Fibrin Conduit with Human Mesenchymal Stem Cells and Immunosuppression after Peripheral Nerve Repair in a Xenogenic Model. Cell Medicine, 10, 1-13
Open this publication in new window or tab >>Long-Term Effects of Fibrin Conduit with Human Mesenchymal Stem Cells and Immunosuppression after Peripheral Nerve Repair in a Xenogenic Model
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2018 (English)In: Cell Medicine, ISSN 2155-1790, Vol. 10, p. 1-13Article in journal (Refereed) Published
Abstract [en]

Introduction: Previously we showed that a fibrin glue conduit with human mesenchymal stem cells (hMSCs) and cyclosporine A (CsA) enhanced early nerve regeneration. In this study long term effects of this conduit are investigated. Methods: In a rat model, the sciatic nerve was repaired with fibrin conduit containing fibrin matrix, fibrin conduit containing fibrin matrix with CsA treatment and fibrin conduit containing fibrin matrix with hMSCs and CsA treatment, and also with nerve graft as control. Results: At 12 weeks 34% of motoneurons of the control group regenerated axons through the fibrin conduit. CsA treatment alone or with hMSCs resulted in axon regeneration of 67% and 64% motoneurons respectively. The gastrocnemius muscle weight was reduced in the conduit with fibrin matrix. The treatment with CsA or CsA with hMSCs induced recovery of the muscle weight and size of fast type fibers towards the levels of the nerve graft group. Discussion: The transplantation of hMSCs for peripheral nerve injury should be optimized to demonstrate their beneficial effects. The CsA may have its own effect on nerve regeneration.

National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-60908 (URN)10.1177/2155179018760327 (DOI)000433910200001 ()
Available from: 2012-11-02 Created: 2012-11-01 Last updated: 2018-09-10Bibliographically approved
Jones, I., Novikova, L. N., Novikov, L. N., Renardy, M., Ullrich, A., Wiberg, M., . . . Kingham, P. J. (2018). Regenerative effects of human embryonic stem cell-derived neural crest cells for treatment of peripheral nerve injury. Journal of Tissue Engineering and Regenerative Medicine, 12(4), E2099-E2109
Open this publication in new window or tab >>Regenerative effects of human embryonic stem cell-derived neural crest cells for treatment of peripheral nerve injury
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2018 (English)In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005, Vol. 12, no 4, p. E2099-E2109Article in journal (Refereed) Published
Abstract [en]

Surgical intervention is the current gold standard treatment following peripheral nerve injury. However, this approach has limitations, and full recovery of both motor and sensory modalities often remains incomplete. The development of artificial nerve grafts that either complement or replace current surgical procedures is therefore of paramount importance. An essential component of artificial grafts is biodegradable conduits and transplanted cells that provide trophic support during the regenerative process. Neural crest cells are promising support cell candidates because they are the parent population to many peripheral nervous system lineages. In this study, neural crest cells were differentiated from human embryonic stem cells. The differentiated cells exhibited typical stellate morphology and protein expression signatures that were comparable with native neural crest. Conditioned media harvested from the differentiated cells contained a range of biologically active trophic factors and was able to stimulate in vitro neurite outgrowth. Differentiated neural crest cells were seeded into a biodegradable nerve conduit, and their regeneration potential was assessed in a rat sciatic nerve injury model. A robust regeneration front was observed across the entire width of the conduit seeded with the differentiated neural crest cells. Moreover, the up-regulation of several regeneration-related genes was observed within the dorsal root ganglion and spinal cord segments harvested from transplanted animals. Our results demonstrate that the differentiated neural crest cells are biologically active and provide trophic support to stimulate peripheral nerve regeneration. Differentiated neural crest cells are therefore promising supporting cell candidates to aid in peripheral nerve repair.

Keywords
artificial nerve graft, human embryonic stem cells, neural crest cells, peripheral nerve injuries, ripheral nervous system, VELOPMENTAL EVOLUTION, V314B, P95 e Gabsang, 2007, NATURE BIOTECHNOLOGY, V25, P1468
National Category
Neurosciences
Identifiers
urn:nbn:se:umu:diva-147466 (URN)10.1002/term.2642 (DOI)000430395400024 ()29327452 (PubMedID)
Funder
Swedish Research Council, 2014-2306
Available from: 2018-05-28 Created: 2018-05-28 Last updated: 2018-06-09Bibliographically approved
Ching, R. C., Wiberg, M. & Kingham, P. J. (2018). Schwann cell-like differentiated adipose stem cells promote neurite outgrowth via secreted exosomes and RNA transfer. Stem Cell Research & Therapy, 9, Article ID 266.
Open this publication in new window or tab >>Schwann cell-like differentiated adipose stem cells promote neurite outgrowth via secreted exosomes and RNA transfer
2018 (English)In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 9, article id 266Article in journal (Refereed) Published
Abstract [en]

Background: Adipose derived stem cells can be stimulated to produce a growth factor rich secretome which enhances axon regeneration. In this study we investigated the importance of exosomes, extracellular vesicles released by many different cell types, including stem cells and endogenous nervous system Schwann cells (SCs), on neurite outgrowth.

Methods: Adipose derived stem cells were differentiated towards a Schwann cell-like phenotype (dADSCs) by in vitro stimulation with a mix of factors (basic fibroblast growth factor, platelet derived growth factor-AA, neuregulin-1 and forskolin). Using a precipitation and low-speed centrifugation protocol the extracellular vesicles were isolated from the medium of the stem cells cultures and also from primary SCs. The conditioned media or concentrated vesicles were applied to neurons in vitro and computerised image analysis was used to assess neurite outgrowth. Total RNA was purified from the extracellular vesicles and investigated using qRT-PCR.

Results: Application of exosomes derived from SCs significantly enhanced in vitro neurite outgrowth and this was replicated by the exosomes from dADSCs. qRT-PCR demonstrated that the exosomes contained mRNAs and miRNAs known to play a role in nerve regeneration and these molecules were up-regulated by the Schwann cell differentiation protocol. Transfer of fluorescently tagged exosomal RNA to neurons was detected and destruction of the RNA by UV-irradiation significantly reduced the dADSCs exosome effects on neurite outgrowth. In contrast, this process had no significant effect on the SCs-derived exosomes.

Conclusions: In summary, this work suggests that stem cell-derived exosomes might be a useful adjunct to other novel therapeutic interventions in nerve repair.

Place, publisher, year, edition, pages
BioMed Central, 2018
Keywords
Exosomes, Extracellular vesicles, Peripheral nerves, Regeneration, Schwann cells, Stem cells
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-153126 (URN)10.1186/s13287-018-1017-8 (DOI)000447275400002 ()30309388 (PubMedID)2-s2.0-85054757041 (Scopus ID)
Available from: 2018-11-09 Created: 2018-11-09 Last updated: 2018-11-09Bibliographically approved
El-Habta, R., Sloniecka, M., Kingham, P. J. & Backman, L. J. (2018). The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts. Stem Cell Research & Therapy, 9, Article ID 352.
Open this publication in new window or tab >>The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts
2018 (English)In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 9, article id 352Article in journal (Refereed) Published
Abstract [en]

Background: Adipose tissue is an excellent source for isolation of stem cells for treating various clinical conditions including injuries to the neuromuscular system. Many previous studies have focused on differentiating these adipose stem cells (ASCs) towards a Schwann cell-like phenotype (dASCs), which can enhance axon regeneration and reduce muscle atrophy. However, the stromal vascular fraction (SVF), from which the ASCs are derived, also exerts broad regenerative potential and might provide a faster route to clinical translation of the cell therapies for treatment of neuromuscular disorders.

Methods: The aim of this study was to establish the effects of SVF cells on the proliferation and differentiation of myoblasts using indirect co-culture experiments. A Growth Factor PCR Array was used to compare the secretomes of SVF and dASCs, and the downstream signaling pathways were investigated.

Results: SVF cells, unlike culture-expanded dASCs, expressed and secreted hepatocyte growth factor (HGF) at concentrations sufficient to enhance the proliferation of myoblasts. Pharmacological inhibitor studies revealed that the signal is mediated via ERK1/2 phosphorylation and that the effect is significantly reduced by the addition of 100 pM Norleual, a specific HGF inhibitor. When myoblasts were differentiated into multinucleated myotubes, the SVF cells reduced the expression levels of fast-type myosin heavy chain (MyHC2) suggesting an inhibition of the differentiation process.

Conclusions: In summary, this study shows the importance of HGF as a mediator of the SVF effects on myoblasts and provides further evidence for the importance of the secretome in cell therapy and regenerative medicine applications.

Place, publisher, year, edition, pages
BioMed Central, 2018
Keywords
Adipose stem cells, Differentiation, HGF, Myoblasts, Myotubes, Myogenesis, Proliferation, SVF
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-155097 (URN)10.1186/s13287-018-1096-6 (DOI)000454165400001 ()30572954 (PubMedID)
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2019-01-10Bibliographically approved
Novikova, L. N., Kolar, M. K., Kingham, P. J., Ullrich, A., Oberhoffner, S., Renardy, M., . . . Novikov, L. N. (2018). Trimethylene carbonate-caprolactone conduit with poly-p-dioxanone microfilaments to promote regeneration after spinal cord injury. Acta Biomaterialia, 66, 177-191
Open this publication in new window or tab >>Trimethylene carbonate-caprolactone conduit with poly-p-dioxanone microfilaments to promote regeneration after spinal cord injury
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2018 (English)In: Acta Biomaterialia, ISSN 1742-7061, E-ISSN 1878-7568, Vol. 66, p. 177-191Article in journal (Refereed) Published
Abstract [en]

Spinal cord injury (SCI) is often associated with scarring and cavity formation and therefore bridging strategies are essential to provide a physical substrate for axonal regeneration. In this study we investigated the effects of a biodegradable conduit made from trimethylene carbonate and c-caprolactone (TC) containing poly-p-dioxanone microfilaments (PDO) with longitudinal grooves on regeneration after SCI in adult rats. In vitro studies demonstrated that different cell types including astrocytes, meningeal fibroblasts, Schwann cells and adult sensory dorsal root ganglia neurons can grow on the TC and PDO material. For in vivo experiments, the TC/PDO conduit was implanted into a small 2-3 mm long cavity in the C3-C4 cervical segments immediately after injury (acute SCI) or at 2-5 months after initial surgery (chronic SCI). At 8 weeks after implantation into acute SCI, numerous 5HT-positive descending raphaespinal axons and sensory CGRP-positive axons regenerated across the conduit and were often associated with PDO microfilaments and migrated host cells. Implantation into chronically injured SCI induced regeneration mainly of the sensory CGRP-positive axons. Although the conduit had no effect on the density of OX42-positive microglial cells when compared with SCI control, the activity of GFAP-positive astrocytes was reduced. The results suggest that a TC/PDO conduit can support axonal regeneration after acute and chronic SCI even without addition of exogenous glial or stem cells.

Place, publisher, year, edition, pages
Elsevier, 2018
Keywords
Synthetic conduit, Tissue engineering, Biomaterials, Spinal cord injury, Regeneration
National Category
Biomaterials Science
Identifiers
urn:nbn:se:umu:diva-145385 (URN)10.1016/j.actbio.2017.11.028 (DOI)000424309200013 ()29174588 (PubMedID)
Available from: 2018-03-01 Created: 2018-03-01 Last updated: 2018-06-09Bibliographically approved
Wiberg, R., Kingham, P. J. & Novikova, L. (2017). A Morphological and Molecular Characterization of the Spinal Cord after Ventral Root Avulsion or Distal Peripheral Nerve Axotomy Injuries in Adult Rats. Journal of Neurotrauma, 34(3), 652-660
Open this publication in new window or tab >>A Morphological and Molecular Characterization of the Spinal Cord after Ventral Root Avulsion or Distal Peripheral Nerve Axotomy Injuries in Adult Rats
2017 (English)In: Journal of Neurotrauma, ISSN 0897-7151, E-ISSN 1557-9042, Vol. 34, no 3, p. 652-660Article in journal (Refereed) Published
Abstract [en]

Retrograde cell death in sensory dorsal root ganglion cells following peripheral nerve injury is well established. However, available data regarding the underlying mechanism behind injury induced motoneuron death are conflicting. By comparing morphological and molecular changes in spinal motoneurons after L4-L5 ventral root avulsion (VRA) and distal peripheral nerve axotomy (PNA) 7 and 14 days postoperatively, we aimed to gain more insight about the mechanism behind injury-induced motoneuron degeneration. Morphological changes in spinal cord were assessed by using quantitative immunohistochemistry. Neuronal degeneration was revealed by decreased immunostaining for microtubuleassociated protein-2 in dendrites and synaptophysin in presynaptic boutons after both VRA and PNA. Significant motoneuron atrophy was already observed at 7 days post-injury, independently of injury type. Immunostaining for ED1 reactive microglia was significantly elevated in all experimental groups, as well as the astroglial marker glial fibrillary acidic protein (GFAP). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the ventral horn from L4-L5 spinal cord segments revealed a significant upregulation of genes involved in programmed cell death including caspase-3, caspase-8, and related death receptors TRAIL-R, tumor necrosis factor (TNF)-R, and Fas following VRA. In contrast, following PNA, caspase-3 and the death receptor gene expression levels did not differ from the control, and there was only a modest increased expression of caspase-8. Moreover, the altered gene expression correlated with protein changes. These results show that the spinal motoneurons reacted in a similar fashion with respect to morphological changes after both proximal and distal injury. However, the increased expression of caspase-3, caspase-8, and related death receptors after VRA suggest that injury- induced motoneuron degeneration is mediated through an apoptotic mechanism, which might involve both the intrinsic and the extrinsic pathways.

Place, publisher, year, edition, pages
Mary Ann Liebert, 2017
Keywords
apoptosis, motoneurons, PNA, VRA
National Category
Surgery Neurosciences Neurology
Identifiers
urn:nbn:se:umu:diva-127355 (URN)10.1089/neu.2015.4378 (DOI)000392815600013 ()27297543 (PubMedID)
Note

Online Ahead of Print: July 8, 2016

Available from: 2016-11-09 Created: 2016-11-09 Last updated: 2018-06-09Bibliographically approved
Lauvrud, A. T., Kelk, P., Wiberg, M. & Kingham, P. J. (2017). Characterization of human adipose tissue-derived stem cells with enhanced angiogenic and adipogenic properties. Journal of Tissue Engineering and Regenerative Medicine, 11(9), 2490-2502
Open this publication in new window or tab >>Characterization of human adipose tissue-derived stem cells with enhanced angiogenic and adipogenic properties
2017 (English)In: Journal of Tissue Engineering and Regenerative Medicine, ISSN 1932-6254, E-ISSN 1932-7005, Vol. 11, no 9, p. 2490-2502Article in journal (Refereed) Published
Abstract [en]

Autologous fat grafting is a popular method for soft tissue reconstructions but graft survival remains highly unpredictable. Supplementation of the graft with the stromal vascular fraction (SVF) or cultured adipose tissue-derived stem cells (ASCs) can enhance graft viability. In this study we have examined the phenotypic properties of a selected population of cells isolated from ASCs, with a view to determining their suitability for transplantation into grafts. ASCs were isolated from the SVF of human abdominal fat (n = 8 female patients) and CD146(+) cells were selected using immunomagnetic beads. The angiogenic and adipogenic properties of the positively selected cells were compared with the negative fraction. CD146(+) cells expressed the immunophenotypic characteristics of pericytes. With prolonged in vitro expansion, CD146(-) cells exhibited increased population doubling times and morphological signs of senescence, whereas CD146(+) cells did not. CD146(+) cells expressed higher levels of the angiogenic molecules VEGF-A, angiopoietin-1 and FGF-1. Conditioned medium taken from CD146(+) cells significantly increased formation of in vitro endothelial cell tube networks, whereas CD146(-) cells did not. CD146(+) cells could be differentiated into adipocytes in greater numbers than CD146(-) cells. Consistent with this, differentiated CD146(+) cells expressed higher levels of the adipocyte markers adiponectin and leptin. These results suggest that CD146(+) cells selected from a heterogeneous mix of ASCs have more favourable angiogenic and adipogenic properties, which might provide significant benefits for reconstructive and tissue-engineering applications. Copyright © 2016 John Wiley & Sons, Ltd.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
Keywords
Adipogenesis, adult stem cell, angiogenesis, fat grafting, pericyte, regeneration
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-133355 (URN)10.1002/term.2147 (DOI)000411481600006 ()26833948 (PubMedID)
Available from: 2017-04-06 Created: 2017-04-06 Last updated: 2018-06-09Bibliographically approved
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ORCID iD: ORCID iD iconorcid.org/0000-0003-2596-5936

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