Dataset Information

CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration.

ABSTRACT: Background: Peripheral nerve regeneration requires coordinated functions of neurotrophic factors and neuronal cells. CRISPR activation (CRISPRa) is a powerful tool that exploits inactive Cas9 (dCas9), single guide RNA (sgRNA) and transcription activator for gene activation, but has yet to be harnessed for tissue regeneration. Methods: We developed a hybrid baculovirus (BV) vector to harbor and deliver the CRISPRa system for multiplexed activation of 3 neurotrophic factor genes (BDNF, GDNF and NGF). The hybrid BV was used to transduce rat adipose-derived stem cells (ASC) and functionalize the ASC sheets. We further implanted the ASC sheets into sciatic nerve injury sites in rats. Results: Transduction of rat ASC with the hybrid BV vector enabled robust, simultaneous and prolonged activation of the 3 neurotrophic factors for at least 21 days. The CRISPRa-engineered ASC sheets were able to promote Schwann cell (SC) migration, neuron proliferation and neurite outgrowth in vitro. The CRISPRa-engineered ASC sheets further enhanced in vivo functional recovery, nerve reinnervation, axon regeneration and remyelination. Conclusion: These data collectively implicated the potentials of the hybrid BV-delivered CRISPRa system for multiplexed activation of endogenous neurotrophic factor genes in ASC sheets to promote peripheral nerve regeneration.

SUBMITTER: Hsu MN

PROVIDER: S-EPMC6735509 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration.

Hsu Mu-Nung MN Liao Han-Tsung HT Truong Vu Anh VA Huang Kai-Lun KL Yu Fu-Jen FJ Chen Hwei-Hsien HH Nguyen Thi Kieu Nuong TKN Makarevich Pavel P Parfyonova Yelena Y Hu Yu-Chen YC

Theranostics 20190814 21

Background: Peripheral nerve regeneration requires coordinated functions of neurotrophic factors and neuronal cells. CRISPR activation (CRISPRa) is a powerful tool that exploits inactive Cas9 (dCas9), single guide RNA (sgRNA) and transcription activator for gene activation, but has yet to be harnessed for tissue regeneration. Methods: We developed a hybrid baculovirus (BV) vector to harbor and deliver the CRISPRa system for multiplexed activation of 3 neurotrophic factor genes ( ...[more]

PMID: 31534539

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Project description:Despite microsurgical repair, recovery of function following peripheral nerve injury is slow and often incomplete. Outcomes could be improved by an increased understanding of the molecular biology of regeneration and by translation of experimental bioengineering strategies. Topographical cues have been shown to be powerful regulators of the rate and directionality of neurite regeneration, and in this study we investigated the downstream molecular effects of linear micropatterned structures in an organotypic explant model. Linear topographical cues enhanced neurite outgrowth and our results demonstrated that the mTOR pathway is important in regulating these responses. mTOR gene expression peaked between 48 and 72h, coincident with the onset of rapid neurite outgrowth and glial migration, and correlated with neurite length at 48h. mTOR protein was located to glia and in a punctate distribution along neurites. mTOR levels peaked at 72h and were significantly increased by patterned topography (p<0.05). Furthermore, the topographical cues could override pharmacological inhibition. Downstream phosphorylation assays and inhibition of mTORC1 using rapamycin highlighted mTORC2 as an important mediator, and more specific therapeutic target. Quantitative immunohistochemistry confirmed the presence of the mTORC2 component rictor at the regenerating front where it co-localised with F-actin and vinculin. Collectively, these results provide a deeper understanding of the mechanism of action of topography on neural regeneration, and support the incorporation of topographical patterning in combination with pharmacological mTORC2 potentiation within biomaterial constructs used to repair peripheral nerves. STATEMENT OF SIGNIFICANCE:Peripheral nerve injury is common and functionally devastating. Despite microsurgical repair, healing is slow and incomplete, with lasting functional deficit. There is a clear need to translate bioengineering approaches and increase our knowledge of the molecular processes controlling nerve regeneration to improve the rate and success of healing. Topographical cues are powerful determinants of neurite outgrowth and represent a highly translatable engineering strategy. Here we demonstrate, for the first time, that microtopography potentiates neurite outgrowth via the mTOR pathway, with the mTORC2 subtype being of particular importance. These results give further evidence for the incorporation of microtopographical cues into peripheral nerve regeneration conduits and indicate that mTORC2 may be a suitable therapeutic target to potentiate nerve regeneration.

Project description:BackgroundEfficient and stable delivery of neurotrophic factors (NTFs) is crucial to provide suitable microenvironment for peripheral nerve regeneration. Neurotrophin-3 (NT-3) is an important NTF during peripheral nerve regeneration which is scarce in the first few weeks of nerve defect. Exosomes are nanovesicles and have been served as promising candidate for biocarrier. In this work, NT-3 mRNA was encapsulated in adipose-derived stem cell (ADSC)-derived exosomes (ExoNT-3). These engineered exosomes were applied as NT-3 mRNA carrier and then were loaded in nerve guidance conduit (ExoNT-3-NGC) to bridge rat sciatic nerve defect.MethodNT-3 mRNA was encapsulated in exosomes by forcedly expression of NT-3 mRNA in the donor ADSCs. ExoNT-3 were co-cultured with SCs in vitro; after 24 h of culture, the efficiency of NT-3 mRNA delivery was evaluated by qPCR, western blotting and ELISA. Then, ExoNT-3 were loaded in alginate hydrogel to construct the nerve guidance conduits (ExoNT-3-NGC). ExoNT-3-NGC were implanted in vivo to reconstruct 10 mm rat sciatic nerve defect. The expression of NT-3 was measured 2 weeks after the implantation operation. The sciatic nerve functional index (SFI) was examined at 2 and 8 weeks after the operation. Moreover, the therapeutic effect of ExoNT-3-NGC was also evaluated by morphology assay, immunofluorescence staining of regenerated nerves, function evaluation of gastrocnemius muscles after 8 weeks of implantation.ResultsThe engineered exosomes could deliver NT-3 mRNA to the recipient cells efficiently and translated into functional protein. The constructed NGC could realize stable release of exosomes at least for 2 weeks. After NGC implantation in vivo, ExoNT-3-NGC group significantly promote nerve regeneration and improve the function recovery of gastrocnemius muscles compared with control exosomes (Exoempty-NGC) group.ConclusionIn this work, NGC was constructed to allow exosome-mediated NT-3 mRNA delivery. After ExoNT-3-NGC implantation in vivo, the level of NT-3 could restore which enhance the nerve regeneration. Our study provide a potential approach to improve nerve regeneration.

Project description:The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor (GDNF) has been shown to protect neurons by binding to N-cadherin. Our previous results have shown that electroacupuncture could increase the expression of N-cadherin mRNA in facial neurons and promote facial nerve regeneration. In this study, the potential mechanisms by which electroacupuncture promotes nerve regeneration were elucidated through assessing the effects of electroacupuncture on GDNF and N-cadherin expression in facial motoneurons of rabbits with peripheral facial nerve crush injury. New Zealand rabbits were randomly divided into a normal group (normal control, n = 21), injury group (n = 45) and electroacupuncture group (n = 45). Model rabbits underwent facial nerve crush injury only. Rabbits in the electroacupuncture group received facial nerve injury, and then underwent electroacupuncture at Yifeng (TE17), Jiache (ST6), Sibai (ST2), Dicang (ST4), Yangbai (GB14), Quanliao (SI18), and Hegu (LI4; only acupuncture, no electrical stimulation). The results showed that in behavioral assessments, the total scores of blink reflex, vibrissae movement, and position of apex nasi, were markedly lower in the EA group than those in the injury group. Hematoxylin-eosin staining of the right buccinator muscle of each group showed that the cross-sectional area of buccinator was larger in the electroacupuncture group than in the injury group on days 1, 14 and 21 post-surgery. Toluidine blue staining of the right facial nerve tissue of each group revealed that on day 14 post-surgery, there was less axonal demyelination and fewer inflammatory cells in the electroacupuncture group compared with the injury group. Quantitative real time-polymerase chain reaction showed that compared with the injury group, N-cadherin mRNA levels on days 4, 7, 14 and 21 and GDNF mRNA levels on days 4, 7 and 14 were significantly higher in the electroacupuncture group. Western blot assay displayed that compared with the injury group, the expression of GDNF protein levels on days 7, 14 and 21 were significantly upregulated in the electroacupuncture group. The histology with hematoxylin-eosin staining and Nissl staining of brainstem tissues containing facial neurons in the middle and lower part of the pons exhibited that on day 7 post-surgery, there were significantly fewer apoptotic neurons in the electroacupuncture group than in the injury group. By day 21, there was no significantly difference in the number of neurons between the electroacupuncture and normal groups. Taken together, these results have confirmed that electroacupuncture promotes regeneration of peripheral facial nerve injury in rabbits, inhibits neuronal apoptosis, and reduces peripheral inflammatory response, resulting in the recovery of facial muscle function. This is achieved by up-regulating the expression of GDNF and N-cadherin in central facial neurons.

Dataset Information

CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration.

Publications

CRISPR-based Activation of Endogenous Neurotrophic Genes in Adipose Stem Cell Sheets to Stimulate Peripheral Nerve Regeneration.

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