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Cell therapy for denervated tissue
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy.ORCID iD: 0000-0001-5225-0595
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Background: Peripheral nerve injury results in denervation of tendons and muscles. The biology of denervated muscle has been well studied but little is known about the associated tendons. Denervation of muscle leads to atrophy which includes muscle fiber shrinkage and cell death, a process that is influenced by the lack of acetylcholine (ACh) signaling to the muscle cells. Recovery of long-term denervated muscle function is often poor. This thesis describes how a cell therapy approach using adipose tissue-derived stromal vascular fraction (SVF) may be used to protect and regenerate denervated muscle. Previous studies have shown how adipose tissue-dervied stem cells (ASCs), commonly expanded from the SVF, have pro-regenerative effects on the injured peripheral nervous system, and how ASCs differentiated towards a “Schwann cell-like phenotype” (dASCs) reduce muscle atrophy. In this thesis work, we studied the possible mechanisms underlying the regenerative potential of both SVF and culture expanded dASCs.

Hypotheses: We hypothesized that: 1) denervated tendon displays morphological and biochemical properties that resemble the chronic degenerative tendon condition known as tendinosis; 2) denervated muscle up-regulates expression of muscarinic acetylcholine (ACh) receptors and apoptosis-associated signaling mechanisms; 3) dASCs enhance the proliferation of myoblasts in vitro through secretion of ACh; 4) SVF influences the proliferation, differentiation, and survival of myoblasts in vitro via secretion of growth factors; and 5) SVF can preserve denervated muscle tissue. To test our hypotheses, two model systems were used: an in vitro model based on indirect co-culture, and an in vivo rat sciatic nerve transection model.

Results: Denervated tendon displayed morphological changes similar to tendinosis, including hypercellularity, disfigurement of cells, and disorganized collagen architecture, along with an increased expression of type I and type III collagen. In addition, levels of neurokinin 1 receptor (NK-1R) were upregulated in the tendon cells. In denervated muscle, there was an increased expression of muscarinic ACh receptors, as well as of genes associated with apoptosis, such as caspases, cytokines (e.g., tumor necrosis factor-alpha; TNF-a), and death domain receptors. We subsequently used TNF-aas an inducer of apoptosis in an in vitrorat primary myoblast culture model. TNF-aactivated/cleaved caspase 7 and increased poly ADP-ribose polymerase (PARP) levels. Moreover, Annexin V and TUNEL were increased after TNF-atreatment. Indirect co-culture with SVF significantly reduced all these measures of apoptosis. Proliferation studies showed that both dASCs and SVF enhanced growth of myoblasts in vitro. With dASCs, the effect was partially explained by secretion of ACh, and for SVF by released growth factors, such as hepatocyte growth factor (HGF). In both cases, the signal was mediated via phosphorylation of ERK1/2 (MAPK). HGF also had an inhibitory effect on the differentiation of myoblasts into myotubes. Finally, the protective effects of SVF were confirmed in vivo: injections of SVF into denervated muscle significantly increased the mean fiber area and diameter, as well as reduced the expression of apoptotic genes and TUNEL reactivity.

ConclusionsDenervated tendons undergo severe degenerative changes similar to tendinosis. Furthermore, SVF has the ability to reduce muscle atrophy in vivo. Using in vitro systems, we showed that this might occur through secretion of growth factors which activate MAPK signaling and anti-apoptotic pathways. In conclusion, SVF offers a promising approach for future clinical application in the treatment of denervated muscle.

Place, publisher, year, edition, pages
Umeå: Umeå universitet , 2020. , p. 91
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2077
Keywords [en]
adipose tissue-derived stem cell, cell therapy, denervation, differentiation, myoblast, peripheral nerve injury, proliferation, regeneration, skeletal muscle, stromal vascular fraction, survival, tendon
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
cell research; Medical Cell Biology
Identifiers
URN: urn:nbn:se:umu:diva-169793ISBN: 978-91-7855-228-3 (print)ISBN: 978-91-7855-229-0 (electronic)OAI: oai:DiVA.org:umu-169793DiVA, id: diva2:1424875
Public defence
2020-05-14, Medicinskt Biologiskt Centrum, Aula Biologica, Umeå, 13:00 (English)
Opponent
Supervisors
Note

ISBN för elektronisk version saknas i fulltexten.

Available from: 2020-04-23 Created: 2020-04-20 Last updated: 2020-04-21Bibliographically approved
List of papers
1. Tendinosis-like changes in denervated rat Achilles tendon
Open this publication in new window or tab >>Tendinosis-like changes in denervated rat Achilles tendon
2018 (English)In: BMC Musculoskeletal Disorders, E-ISSN 1471-2474, Vol. 19, article id 426Article in journal (Refereed) Published
Abstract [en]

Background: Tendon disorders are common and lead to significant disability and pain. Our knowledge of the ‘tennis elbow’, the ‘jumpers knee’, and Achilles tendinosis has increased over the years, but changes in denervated tendons is yet to be described in detail. The aim of the present study was to investigate the morphological and biochemical changes in tendon tissue following two weeks of denervation using a unilateral sciatic nerve transection model in rat Achilles tendons.

Methods: Tendons were compared with respect to cell number, nuclear roundness, and fiber structure. The non-denervated contralateral tendon served as a control. Also, the expression of neuromodulators such as substance P and its preferred receptor neurokinin-1 receptor, NK-1R, was evaluated using real-time qRT-PCR.

Results: Our results showed that denervated tendons expressed morphological changes such as hypercellularity; disfigured cells; disorganization of the collagen network; increased production of type III collagen; and increased expression of NK-1R.

Conclusion: Taken together these data provide new insights into the histopathology of denervated tendons showing that denervation causes somewhat similar changes in the Achilles tendon as does tendinosis in rats.

Place, publisher, year, edition, pages
BMC, 2018
Keywords
Collagen, Denervation, Rat, Substance P, Tendinosis
National Category
Orthopaedics
Identifiers
urn:nbn:se:umu:diva-154940 (URN)10.1186/s12891-018-2353-7 (DOI)000451699800006 ()30497469 (PubMedID)2-s2.0-85057541287 (Scopus ID)
Available from: 2019-01-08 Created: 2019-01-08 Last updated: 2024-01-17Bibliographically approved
2. Adipose stem cells enhance myoblast proliferation via acetylcholine and extracellular signal-regulated kinase 1/2 signaling
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)2-s2.0-85040745017 (Scopus ID)
Available from: 2018-02-08 Created: 2018-02-08 Last updated: 2023-03-24Bibliographically approved
3. The adipose tissue stromal vascular fraction secretome enhances the proliferation but inhibits the differentiation of myoblasts
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)2-s2.0-85058900109 (Scopus ID)
Available from: 2019-01-10 Created: 2019-01-10 Last updated: 2023-03-24Bibliographically approved
4. Anti-apoptotic effect of adipose tissue-derived stromal vascular fraction in denervated rat muscle
Open this publication in new window or tab >>Anti-apoptotic effect of adipose tissue-derived stromal vascular fraction in denervated rat muscle
2021 (English)In: Stem Cell Research & Therapy, E-ISSN 1757-6512, Vol. 12, no 162Article in journal (Refereed) Published
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-169728 (URN)10.1186/s13287-021-02230-y (DOI)000626520900001 ()2-s2.0-85102009955 (Scopus ID)
Available from: 2020-04-20 Created: 2020-04-20 Last updated: 2023-09-05Bibliographically approved

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