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

Hsu, Mu Nung; Liao, Han-Tsung; Truong, Vu Anh; Huang, Kai Lun; Yu, Fu Jen; Chen, Hwei Hsien; Nguyen, Thị Kieu Nuong; Макаревич, П.И.; Parfyonova, Yelena; Hu, Yu

doi:10.7150/thno.36790

Cited by 42 publications

(29 citation statements)

References 48 publications

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“…Peripheral nerve injury is one of the most frequently encountered clinical settings resulting in morbidity and lifelong disability and poses a considerable burden for both the individual and the society 1 , 2 . Despite the progress in surgical intervention, the outcome of peripheral nerve injury remains unsatisfactory 3 , 4 .…”

Section: Introductionmentioning

confidence: 99%

Mechanical stimulation of Schwann cells promote peripheral nerve regeneration via extracellular vesicle-mediated transfer of microRNA 23b-3p

Xia¹,

Gao²,

Li³

et al. 2020

Theranostics

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Rationale: Peripheral nerves are unique in their remarkable elasticity. Schwann cells (SCs), important components of the peripheral nervous system (PNS), are constantly subjected to physiological and mechanical stresses from dynamic stretching and compression forces during movement. So far, it is not clear if SCs sense and respond to mechanical signals. It is also unknown whether mechanical stimuli can interfere with the intercellular communications between neurons and SCs, and what role extracellular vesicles (EVs) play in this process. The present study aimed to examine the effect of mechanical stimuli on the EV-mediated intercellular communication between neurons and SCs, explore their effect on axonal regeneration, and investigate the underlying mechanism. Methods: Purified SCs were stimulated using a magnetic force-based mechanical stimulation (MS) system and EVs were purified from mechanically stimulated SCs (MS-SCs-EVs) and non-stimulated SCs (SCs-EVs). The effect of MS-SCs-EVs on axonal elongation was examined in vitro and in vivo . High throughput miRNA sequencing was performed to compare the differential miRNA profiles between MS-SCs-EVs and SCs-EVs. The functional role of differentially expressed miRNAs on neurite extension in MS-SCs-EVs was examined. Also, the putative target genes of differentially expressed miRNAs in MS-SCs-EVs were predicted by bioinformatics tools, and the regulatory effect of those miRNAs on putative target genes was validated both in vitro and in vivo . Results: The MS-SCs-EVs showed an average size of 137.52±1.77 nm, and could be internalized by dorsal root ganglion (DRG) neurons. Compared to SCs-EVs, MS-SCs-EVs showed a stronger ability to enhance neurite outgrowth in vitro and nerve regeneration in vivo . High throughput miRNA sequencing identified a number of differentially expressed miRNAs in MS-SCs-EVs. Further analysis of those EV-miRNAs demonstrated that miR-23b-3p played a predominant role in MS-SCs-EVs since its deprivation abolished their enhanced axonal elongation. Furthermore, we identified neuropilin 1 (Nrp1) in neurons as the target gene of miR-23b-3p in MS-SCs-EVs. This observation was supported by the evidence that miR-23b-3p could decrease Nrp1-3'-UTR-WT luciferase activity in vitro and down-regulate Nrp1 expression in neurons. Conclusion: Our findings suggested that mechanical stimuli are capable of modulating the intercellular communication between neurons and SCs by altering miRNA composition in MS-SCs-EVs. Transfer of miR-23b-3p by MS-SCs-EVs from mechanically stimulated SCs to neurons decreased neuronal Nrp1 expression, which was responsible, at least in part, for the beneficial effect of MS-SCs-EVs on axonal regeneration. Our results highlighted the potential therapeutic value of MS-SCs-EVs and miR-23b-3p-en...

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Section: Introductionmentioning

confidence: 99%

Mechanical stimulation of Schwann cells promote peripheral nerve regeneration via extracellular vesicle-mediated transfer of microRNA 23b-3p

Xia¹,

Gao²,

Li³

et al. 2020

Theranostics

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“…The functional peptide motif IKVAV is commonly used in neurotrophin releasing and stem cell loading during treatment of traumatic brain injury, spinal cord injury, and sciatic nerve injury due to its ability to enhance cell survival and adhesion, neurite outgrowth, and even angiogenesis 25 - 27 . BDNF is a member of the neurotrophin family and enhances myelination in SCs, promotes neuronal survival and neurite outgrowth, and prevents neural death 28 , 29 . Studies indicate that BDNF promotes axonal regrowth after sciatic nerve injury and is closely related to motor recovery following PNI 30 - 33 .…”

Section: Introductionmentioning

confidence: 99%

Self-assembling peptide hydrogels functionalized with LN- and BDNF- mimicking epitopes synergistically enhance peripheral nerve regeneration

et al. 2020

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The regenerative capacity of the peripheral nervous system is closely related to the role that Schwann cells (SCs) play in construction of the basement membrane containing multiple extracellular matrix proteins and secretion of neurotrophic factors, including laminin (LN) and brain-derived neurotrophic factor (BDNF). Here, we developed a self-assembling peptide (SAP) nanofiber hydrogel based on self-assembling backbone Ac-(RADA) 4 -NH 2 (RAD) dual-functionalized with laminin-derived motif IKVAV (IKV) and a BDNF-mimetic peptide epitope RGIDKRHWNSQ (RGI) for peripheral nerve regeneration, with the hydrogel providing a three-dimensional (3D) microenvironment for SCs and neurites. Methods: Circular dichroism (CD), atomic force microscopy (AFM), and scanning electron microscopy (SEM) were used to characterize the secondary structures, microscopic structures, and morphologies of self-assembling nanofiber hydrogels. Then the SC adhesion, myelination and neurotrophin secretion were evaluated on the hydrogels. Finally, the SAP hydrogels were injected into hollow chitosan tubes to bridge a 10-mm-long sciatic nerve defect in rats, and in vivo gene expression at 1 week, axonal regeneration, target muscular re-innervation, and functional recovery at 12 weeks were assessed. Results: The bioactive peptide motifs were covalently linked to the C-terminal of the self-assembling peptide and the functionalized peptides could form well-defined nanofibrous hydrogels capable of providing a 3D microenvironment similar to native extracellular matrix. SCs displayed improved cell adhesion on hydrogels with both IKV and RGI, accompanied by increased cell spreading and elongation relative to other groups. RSCs cultured on hydrogels with IKV and RGI showed enhanced gene expression of NGF, BDNF, CNTF, PMP22 and NRP2, and decreased gene expression of NCAM compared with those cultured on other three groups after a 7-day incubation. Additionally, the secretion of NGF, BDNF, and CNTF of RSCs was significantly improved on dual-functionalized peptide hydrogels after 3 days. At 1 week after implantation, the expressions of neurotrophin and myelin-related genes in the nerve grafts in SAP and Autograft groups were higher than that in Hollow group, and the expression of S100 in groups containing both IKV and RGI was significantly higher than that in groups containing either IKV or RGI hydrogels, suggesting enhanced SC proliferation. The morphometric parameters of the regenerated nerves, their electrophysiological performance, the innervated muscle weight and remodeling of muscle fibers, and motor function showed that RAD/IKV/RGI and RAD/IKV-GG-RGI hydrogels could markedly improve axonal regeneration with enhanced re-myelination and motor functional recovery through the synergetic effect of IKV and RGI functional motifs. Conclusions: We found that the dual-functionalized SAP hydrogels promoted RSC adhesion, my...

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“…Finally, the ability of MSC to form tissue-like constructs composing of viable cells and their native ECM resulted in the development of cell sheets (CS)-minimal tissue-engineered constructs that found application for many conditions. Our previous experience with MSC-based CS showed their efficacy for stimulation of angiogenesis, nerve repair and cardiac regeneration and proved their superiority over routinely used local injection [10][11][12][13][14]. The latter has been shown to induce MSC apoptosis and its rapid clearance from blood flow or tissue (muscle, myocardium, etc.).…”

Section: Introductionmentioning

confidence: 94%

Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization

Александрушкина

Nimiritsky

Eremichev

et al. 2020

IJMS

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We report a comparative study of multipotent mesenchymal stromal cells (MSC) delivered by injection, MSC-based cell sheets (CS) or MSC secretome to induce healing of cutaneous pressure ulcer in C57Bl/6 mice. We found that transplantation of CS from adipose-derived MSC resulted in reduction of fibrosis and recovery of skin structure with its appendages (hair and cutaneous glands). Despite short retention of CS on ulcer surface (3–7 days) it induced profound changes in granulation tissue (GT) structure, increasing its thickness and altering vascularization pattern with reduced blood vessel density and increased maturation of blood vessels. Comparable effects on GT vascularization were induced by MSC secretome, yet this treatment has failed to induce repair of skin with its appendages we observed in the CS group. Study of secretome components produced by MSC in monolayer or sheets revealed that CS produce more factors involved in pericyte chemotaxis and blood vessel maturation (PDGF-BB, HGF, G-CSF) but not sprouting inducer (VEGF165). Analysis of transcriptome using RNA sequencing and Gene Ontology mapping found in CS upregulation of proteins responsible for collagen binding and GT maturation as well as fatty acid metabolism enzymes known to be negative regulators of blood vessel sprouting. At the same time, downregulated transcripts were enriched by factors activating capillary growth, suggesting that in MSC sheets paracrine activity may shift towards matrix remodeling and maturation of vasculature, but not activation of blood vessel sprouting. We proposed a putative paracrine trigger mechanism potentially rendering an impact on GT vascularization and remodeling. Our results suggest that within sheets, MSC may change their functional state and spectrum of soluble factors that influence tissue repair and induce more effective skin healing inclining towards regeneration and reduced scarring.

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

Cited by 42 publications

References 48 publications

Mechanical stimulation of Schwann cells promote peripheral nerve regeneration via extracellular vesicle-mediated transfer of microRNA 23b-3p

Mechanical stimulation of Schwann cells promote peripheral nerve regeneration via extracellular vesicle-mediated transfer of microRNA 23b-3p

Self-assembling peptide hydrogels functionalized with LN- and BDNF- mimicking epitopes synergistically enhance peripheral nerve regeneration

Cell Sheets from Adipose Tissue MSC Induce Healing of Pressure Ulcer and Prevent Fibrosis via Trigger Effects on Granulation Tissue Growth and Vascularization

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